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).

scholarly journals A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle

2019 ◽  
Vol 31 (12) ◽  
pp. 1793 ◽  
Author(s):  
Valentina Lodde ◽  
Silvia Colleoni ◽  
Irene Tessaro ◽  
Davide Corbani ◽  
Giovanna Lazzari ◽  
...  
Keyword(s):  
Gap Junction ◽  
Embryo Development ◽  
Large Scale ◽  
Germinal Vesicle ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Morphological Criteria ◽  
Chromatin Configuration ◽  
Follicle Size ◽  
Meiotic Competence

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus–oocyte complexes (COCs) according to follicle size (&lt;1, 1–2, &gt;2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles &lt;1 and 1–2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1–2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P&lt;0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles &lt;1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

scholarly journals Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Hsiao Yun Yang ◽  
Shae-Lee Cox ◽  
Graham Jenkin ◽  
Jock Findlay ◽  
Alan Trounson ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Developmental Potential ◽  
Oocyte Yield ◽  
Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

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

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.

Cycloheximide speeds the porcine oocyte nuclear kinetics and retains embryonic developmental potential in the presence of gonadotrophins

2010 ◽  
Vol 90 (2) ◽  
pp. 189-196
Author(s):  
X -L. Sun ◽  
W -Z. Ma ◽  
Y -B. Zhu ◽  
Z -H. Wu ◽  
L. An ◽  
...  
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Electrical Activation ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Developmental Potential ◽  
Oocyte Meiosis

Animal embryo engineering requires large amounts of synchronized mature oocytes in vitro. However, porcine cumulus-oocyte complexes aspirated from 3-8 mm follicles are at different germinal vesicle stages. They reach metaphase II stages asynchronously when cultured in vitro. In this study, we examined the effects of pretreatment with or without cycloheximide (CHX), equine chorionic gonadotrophin (eCG), human chorionic gonadotrophin (hCG), and their combinations on meiotic synchronization and the developmental competence of porcine oocytes in vitro following electrical activation. The COCs were pretreated for 12 h with either control medium (TCM 199), CHX (TCM 199 + CHX), eCG/hCG (TCM 199 + eCG/hCG) or eCG/hCG + CHX (TCM 199 + CHX + eCG/hCG), and then cultured for up to 32 h with TCM199 + eCG/hCG. After 12 h pretreatment, the rates of germinal vesicle breakdown (GVBD) were lower (P < 0.05) in the CHX (8.4%) and eCG/hCG + CHX (1.5%) groups compared with control (55.4%) and eCG/hCG (27.2%) groups. After removal of CHX and culture for an additional 12 h in vitro, the majority of the oocytes were synchronized at the GVBD stage in CHX (75.6%) and eCG/hCG + CHX (65.0%) groups. At additional 32 h of culture, the rate of oocytes in metaphase II in eCG/hCG + CHX group (68.3%) was significantly (P < 0.05) higher than the eCG/hCG group (54.8%), but did not differ from other groups (control: 61.3%, CHX: 58.8%). After electrical activation, the cleavage and blastocyst formation rates in the CHX group (80.3%; 19.5%) were significantly (P < 0.05) lower than those in the control group (95.5%; 45.3%), while no difference was found between eCG/hCG + CHX (82.2%; 34.4%) and control groups. Our data, hence, demonstrate pretreatment with CHX hastened nuclear kinetics of porcine oocytes cultured in vitro; however, embryo development potential was retained only when gonadotrophins is present in the in vitro maturation (IVM) medium. Thus, CHX should be used in the two-step culture systems in combination with gonadotrophins. Key words: Oocyte meiosis, synchronization, cycloheximide, embryo development, pig

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.

266 COMPARISON OF OOCYTES FROM LARGE-SIZED (≥8-mm) and MEDIUM-SIZED (3- to 7-mm) FOLLICLES FOR IN VITRO EMBRYO PRODUCTION IN PIGS

2013 ◽  
Vol 25 (1) ◽  
pp. 281
Author(s):  
J. D. Yoon ◽  
S.-S. Kwak ◽  
Y. Jeong ◽  
S.-A. Jeong ◽  
E. Lee ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Reduced Glutathione ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Nuclear Antigen ◽  
Control Group ◽  
Related Factor ◽  
Parthenogenetic Activation

Despite recent efforts to improve culture systems, the developmental competence of in vitro-matured (IVM) porcine oocytes is still inferior compared with those that have been in vivo matured. In pigs, cumulus–oocyte complexes (COC) are usually aspirated from 3- to 7-mm follicles and matured for 42 to 44 h in vitro. In this study, we compared oocytes obtained from large-sized (≥8-mm) and medium-sized (3- to 7-mm) follicles in terms of nuclear maturation, intracellular reduced glutathione levels, gene expression, and embryo development after IVM. In the control group, COC (n = 521) were aspirated from 3- to 7-mm follicles and matured for 22 h with hormones (eCG/hCG) and subsequently matured in vitro for 20 to 22 h without hormones at 39°C, 5% CO2. In the large follicle (LF) group, COC (n = 256) were obtained from follicles larger than 7 mm and were subjected to IVM reduced for 18 h. The maturation medium was TCM-199 supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng mL–1 of epidermal growth factor, 75 µg mL–1 of kanamycin, 1 µg mL–1 of insulin, and 10% (vol/vol) porcine follicular fluid without hormones. Nuclear status and reduced glutathione content in oocytes were investigated by Hoechst 33342 staining and CellTracker Blue (CMF2HC; Invitrogen, Carlsbad, CA, USA), respectively. The abundance of messenger RNA of genes reflecting the developmental competence of oocytes was analysed in cumulus cells by real-time PCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as the reference. In addition, oocytes were subjected to parthenogenetic activation to assess in vitro embryo developmental competence. Data were analysed by Student’s t-test using SPSS version 17.0 (IBM Corporation, Armonk, NY, USA) and presented as means. All experiments were replicated at least three times. The average frequency of ovaries having ≥8-mm follicles was 2.3% (44/1953 in 11 replicates). Before IVM, the nuclear-stage oocytes from ≥8-mm follicles were as follows: germinal vesicle stage = 15.2%; metaphase I (MI) stage = 55.4%; anaphase I and telophase I (AI + TI) stages = 15.8%; and metaphase II (MII) stage = 13.6%. After 6 h of IVM, 4.2% of oocytes were at the germinal vesicle stage and frequencies of the MI, AI + TI, and MII stages were 43.6, 9.4, and 42.8%, respectively. After 18 h, IVM frequencies of the MI and MII stages were 13.0 and 87.0%, respectively. Oocytes of the LF group showed a significant (P < 0.001) increase in intracellular reduced glutathione level (1.41 v. 1.00) compared with the control (42- to 44-h matured oocytes). Cumulus cells in the LF group showed lower (P < 0.1) messenger RNA expression of COX-2 (cyclooxygenase-2) and TNFAIP6 (tumor necrosis factor, α-induced protein 6), and higher (P < 0.1) expression of PCNA (proliferating cell nuclear antigen) and Nrf2 (NF-E2-related factor 2) compared with the control. After parthenogenetic activation, the oocytes from the LF group had significantly (P < 0.05) higher blastocyst rates and total cell numbers in blastocysts than did the control group (90.1% and 73.6 v. 50.5% and 55.3, respectively). In conclusion, oocytes from preovulatory LF require only 18 h to complete maturation in vitro, and their developmental competence is higher than those obtained from MF. Although limited, oocytes from ≥8-mm follicles offer an alternative source of material for the production of transgenic pigs by somatic cell nuclear transfer. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID

2007 ◽  
Vol 19 (1) ◽  
pp. 268
Author(s):  
A. B. Nascimento ◽  
M. G. Marques ◽  
A. R. de S. Coutinho ◽  
M. N. Tavares ◽  
M. E. O. D'Avila Assumpção ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Penetration Rate ◽  
Early Period ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Control Group ◽  
In Vitro Production

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 �g mL-1), LH (0.5 �g mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 �g mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrode&apos;s buffered medium, mTBM) and placed in petri dishes containing 50 &micro;L of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17&deg;C. It was then centrifuged at 1200g for 3 min and standardized for 1 &times; 105 spermatozoa mL&minus;1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5&deg;C and 5&percnt; CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 &micro;L), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1&percnt; orcein in 45&percnt; acetic acid and evaluated under phase-contrast microscopy at a 400&times; magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 &plusmn; 9.6&percnt; (162/412), and no difference was observed in comparison with the T2 group, 29.5 &plusmn; 4.9&percnt; (113/383). The monospermic penetration rate was 31.5 &plusmn; 6&percnt; (51/162) in the T1 group and differed from that in the T2 group, 71.7 &plusmn; 3.3&percnt; (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 &plusmn; 6&percnt; (111/162), compared with the T2 group, 28.3 &plusmn; 3.3&percnt; (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP &plus; PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.

scholarly journals Effect of the factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro

Reproduction ◽  
2001 ◽  
pp. 249-257 ◽  
Author(s):  
N Isobe ◽  
T Terada
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Conditioned Medium ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Fresh Medium ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion

The present study was undertaken to explore the regulatory mechanisms for meiotic resumption of pig cumulus-oocyte complexes (COCs) by assessing the nuclear status of oocytes, the degree of gap junction cell-to-cell communication and cumulus expansion after culture of various numbers of COCs in 10 microl droplets of medium for 24 h. Gap junction communication was examined by confocal laser scanning microscopy after injection of a fluorescent dye, lucifer yellow, into the oocytes. When one, three or six COCs were cultured in a 10 microl droplet, germinal vesicle breakdown was observed in > 70% of oocytes; increasing the number of COCs in a droplet further actually decreased the proportion of oocytes undergoing germinal vesicle breakdown (10 COCs: 49%; 20 COCs: 21%; 40 COCs: 13%). When six COCs were cultured in a 10 microl droplet of conditioned medium (prepared previously by culturing 20 intact and oocytectomized COCs for 24 h), the proportion of oocytes undergoing germinal vesicle breakdown was significantly reduced compared with the proportion in fresh medium. An increase in the proportion of category 1 COCs (all gap junctions within cumulus cells and between cumulus cells and oocyte are functionally maintained) was achieved by increasing the number of COCs cultured in a 10 microl droplet. The addition of conditioned medium to the fresh medium at a concentration of 50% significantly inhibited cumulus expansion. From these results, it is concluded that the factors secreted by cumulus cells regulate the disruption of gap junctions and cumulus expansion, and concurrently control the incidence of germinal vesicle breakdown in pig COCs.

Sign in / Sign up

Export Citation Format

Share Document