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.

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.

Cortical granule biogenesis is active throughout oogenesis in sea urchins

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1325-1333 ◽  
Author(s):  
M. Laidlaw ◽  
G.M. Wessel
Keyword(s):  
Recombinant Proteins ◽  
Sea Urchins ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Cdna Clones ◽  
Secretory Vesicles ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Peak Abundance ◽  
Cognate Protein

Cortical granules are secretory vesicles formed in the eggs of most animals and are essential for the prevention of polyspermy in these organisms. We have studied the biogenesis of cortical granules in sea urchin oocytes by identifying cDNA clones that encode proteins targeted selectively to the cortical granules. These cDNA clones were identified by an immunoscreen of a cDNA library using antibodies to proteins of the fertilization envelope. Four different mRNAs were identified, ranging from 4 kb to 13 kb in length, that encoded proteins targeted specifically to cortical granules. Accumulation of these mRNAs began very early in oogenesis, in oocytes approximately 10–15 microns in diameter, and continued throughout oogenesis. The mRNAs reached peak abundance (on a per cell basis) in germinal vesicle stage oocytes, and the accumulation of each mRNA was linear with respect to oocyte growth. During breakdown of the germinal vesicle these mRNAs were degraded so that in eggs the mRNA signals were at background levels. Antibodies generated to recombinant proteins made from each of these cDNA clones showed that in the oocyte each cognate protein appeared early in oogenesis. These proteins accumulated only in cortical granules: no accumulation was seen in the cytoplasm, in Golgi, or in other vesicles, and no heterogeneity of the contents was seen within the population of cortical granules. Using these antibodies we show that cortical granules accumulated linearly throughout oogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The localization of LAP2β during pronuclear formation in bovine oocytes after fertilization or activation

Zygote ◽  
2006 ◽  
Vol 14 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Mamiko Isaji ◽  
Hisataka Iwata ◽  
Hiroshi Harayama ◽  
Masashi Miyake
Keyword(s):  
Chromatin Condensation ◽  
Polar Body ◽  
Somatic Cells ◽  
Histone H3 ◽  
Cumulus Cells ◽  
Polar Bodies ◽  
Second Polar Body ◽  
Pronuclear Formation ◽  
Bovine Oocytes

SummaryWe have shown that the assembly of lamin-associated polypeptide (LAP) 2β was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2β assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2β assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2β assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2β assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2β did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2β assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2β assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2β assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2β assembly around oCh but not histone H3 dephosphorylation.

198 CILOSTAMIDE SUSTAINS GAP JUNCTION-MEDIATED COMMUNICATION AND CHROMATIN REMODELLING IN PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
C. Dieci ◽  
F. Franciosi ◽  
V. Lodde ◽  
I. Lagutina ◽  
I. Tessaro ◽  
...  
Keyword(s):  
Gap Junction ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Period ◽  
Control Group ◽  
Functional Coupling ◽  
Mediated Communication ◽  
Developmental Potential ◽  
Chromatin Configuration ◽  
Defined Culture

In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P < 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P < 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

scholarly journals Aspects of cytoplasmic maturation of bovine oocytes: Interplay between mapk, mRNA-cap binding complex and cytoplasmic mRNA metabolism in regulation of translation

2003 ◽  
Vol 19 (3-4) ◽  
pp. 1-8 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Melo Sterza ◽  
M. Kubelka ◽  
Z. Vohnikova ◽  
W. Tomek
Keyword(s):  
Binding Protein ◽  
Germinal Vesicle ◽  
Initiation Factor ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Stage Of Development ◽  
Bovine Oocytes

Bovine oocytes are arrested in the germinal vesicle stage (GV stage)and mature spontaneously when they are removed from their follicles and transferred to a suitable culture medium. This process, known as meiotic maturation is characterized among others, by germinal vesicle breakdown followed by metaphase I (MI) stage and further development to metaphase II (MII), where they become arrested again. During GVBD to MI transition, the overall protein synthesis reaches the highest level and it rapidly declines in MII. We have previously shown that transcription completely declines during meiotic maturation. Therefore we suppose that gene expression is exclusively regulated on translational level at this stage of development. This means that mRNAs, which were stored in repressed form during oocyte growth, were actively translated during meiotic maturation. Therefore we have investigated specific regulators of translation, namely the eukaryotic initiation factor of translation eIF4E (cap binding protein) and a specific repressor of eIF4E function, the 4E-binding protein 4E-BP1. Furthermore, we have elucidated pathways, which lead to eIF4E and 4E-BP1 phosphorylation by using specific M-phase kinase inhibitors, and we compare these results with transcription and cytoplasmic polyadenylation events during the course of meiotic maturation. The detailed knowledge of such regulatory processes can help to improve in vitro bio-techniques and to estimate the risk of these techniques.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Oocyte Growth ◽  
North West ◽  
Significant Difference ◽  
Cytoplasmic Maturation

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

233 DEHIDROLEUCODINE INDUCES PARTHENOGENETIC ACTIVATION OF BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 214
Author(s):  
N. Canel ◽  
D. Salamone
Keyword(s):  
Polar Body ◽  
Metaphase Plate ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Parthenogenetic Activation ◽  
Polar Bodies ◽  
Bovine Oocytes ◽  
Full Activation

Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits germinal vesicle breakdown in Bufo arenarum oocytes. Its action takes place over early stages of the cdc25 activation cascade (Bühler MI et al. 2007 Zygote 15, 183–187). The aim of this study was to evaluate the potential of DhL to induce parthenogenetic activation by observing nuclear dynamics and second polar body (2PB) extrusion of bovine oocytes, in the presence or absence of Cytochalasin B (CB), comparing these treatments with 6-Dimethylaminopurine (DMAP), an activation agent widely used. Cumulus–oocyte complexes were collected from cow ovaries obtained from a slaughterhouse. They were matured in TCM 199, supplemented with 5% FCS, 10 UI mL–1 penicillin, 10 μg mL–1 FSH, 100 μM cysteamine, 0.3 mm sodium pyruvate and 2 mm glutamine, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were selected and treated with 5 μm ionomycin (Io) for 4 min. Afterwards, oocytes were randomly allocated into one of the following treatments: a) incubation with 2 mm DMAP for 3 h (DMAP); b) incubation with 5 μm DhL for 3 h (DhL); and c) incubation with 5 μm DhL and 5 μg mL–1 CB, for 3 h (DhL-CB). A control group was only treated with Io. Activated oocytes were cultured in the maturation medium during 4, 11 or 17 h (Io exposure = 0 h), stained with Hoechst 33342 and analyzed under fluorescence microscope to evaluate nuclear stage and 2PB extrusion. Activation data are presented in Table 1. Oocytes with two extruded polar bodies and a metaphase plate were considered as partially activated (PA) and those exhibiting one pronucleus (PN) or already cleaved, as fully activated (FA). Oocytes that remained arrested at MII were not included in the table. Rates of 2PB emission were 98.3, 4.9, 83.6 and 61.5% for Io, DMAP, DhL and DhL-CB, respectively. These percentages were determined over total number of activated oocytes (PA and FA) within each group, including results from all evaluation times because no differences were found between them. Nuclear evaluation suggests that DhL is as effective as DMAP to induce full activation when combined with CB, and its use does not induce the early PN formation observed with DMAP at 4 h post Io. Most of the oocytes activated with DhL extruded a 2PB; these results were statistically different from those observed for other groups. These results indicate that DhL might be a useful agent to induce parthenogenesis, allowing 2PB extrusion and avoiding early PN formation in bovine oocytes. Table 1.Partial and full activation of bovine oocytes at 4, 11 and 17 h post treatments

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.

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