254. Evidences for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle

2008 ◽  
Vol 20 (9) ◽  
pp. 54
Author(s):  
M. Sasseville ◽  
F. K. Albuz ◽  
F. J. Richard ◽  
R. B. Gilchrist
Keyword(s):  
Ovarian Follicle ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Pde4 Inhibitor ◽  
Bovine Oocyte ◽  
Camp Phosphodiesterase ◽  
Nuclear Maturation ◽  
Activity Measurements ◽  
Camp Levels

3′5’-Cyclic adenosine monophosphate (cAMP) is an important second messenger in the mammalian ovarian follicle implicated in gonadotrophin signalling as well as oocyte meiotic arrest. Cyclic AMP-degrading phosphodiesterases (PDE) modulate cAMP levels in the ovarian follicle, but the specific PDE subtypes responsible for this degradation in the different cellular compartments within the bovine follicle remain unknown. The current dogma, established principally in rodent, presents PDE3A as the ‘oocyte PDE’, while PDE4D is the ‘granulosa/cumulus PDE’. Our PDE activity measurements suggested that a PDE3 (cilostamide-sensitive, 10µM) was representing 79% of the total cAMP-PDE activity in the bovine oocyte, in agreement with the dogma. However, our results suggested that PDE4 (rolipram-sensitive, 10µM) is representing only 19% of the cAMP-PDE activity in the cumulus cells, while 65% of the activity was due to PDE8 (IBMX-insensitive, 500µM), a result in direct opposition with the accepted PDE distribution in the ovarian follicle. Mural granulosa cells were displaying equal amounts of PDE4 (31%) and PDE8 (30%) cAMP-PDE activities. Interestingly, cAMP-PDE activities were not varying during the first 9 h of IVM in the bovine cumulus-oocyte complexes (COC), as seen in rat. COCs treated with an adenylyl cyclase stimulator (forkolin 100µM) in combinaison with the only known inhibitor for the PDE8 family, dipyridamole, are showing a dose-dependant increase of cAMP levels and a significant delay nuclear maturation, whereas a potent PDE4 inhibitor, rolipram (up to 100µM), was ineffective. This study provides the first insight into subtype-specific PDE cAMP degrading activities in the bovine ovarian follicle, especially around oocyte nuclear maturation. It demonstrates dramatic differential PDE subtype compartmentalisation between ovarian somatic cells and the germ cell, including the important contribution of a new PDE family member in the ovarian follicle, PDE8. PDE8 could be a novel pharmacological target to improve bovine oocyte IVM conditions and to increase developmental competence.

79 microRNA EXPRESSION IN BOVINE CUMULUS CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 133
Author(s):  
K. Uhde ◽  
L. T. A. van Tol ◽  
T. A. E. Stout ◽  
B. A. J. Roelen
Keyword(s):  
Mirna Expression ◽  
Reference Genes ◽  
Noncoding Rna ◽  
Ovarian Follicle ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Small Samples ◽  
Variable Expression

A mammalian oocyte within an ovarian follicle is surrounded by cumulus cells, together this structure is known as the cumulus-oocyte complex (COC). Cumulus cells are important for the development of the oocyte, they support the maturation process of the oocyte within the ovary and aid in sperm recognition. Because it is known that a Dicer knockout leads to infertility, microRNAs (miRNA) are focused to have an important role in oocyte development. MiRNAs are small noncoding RNA sequences that act as transcriptional regulators. Little is known about the expression of miRNA in cumulus cells or how cumulus-derived miRNA may regulate or be used to indicate the developmental competence of the maturing oocyte. Our aim was to investigate miRNA expression in oocytes and to identify and establish how specific miRNA influence the acquisition of developmental competence by bovine oocytes. Normalization of qPCR data requires stable reference genes. To this end, we tested the expression of various miRNA with respect to their ability to be used as reference miRNA for bovine cumulus cells; these included miR-103, miR-93, miR-26, let-7a, miR-191, and the small noncoding nuclear RNA U6. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered cows and matured in vitro. Small samples of cumulus cells were collected from these COC before and after maturation. From the cumulus cell groups recovered at different stages, small RNA were extracted and cDNA was synthesised, followed by qRT-PCR. To identify the optimal combination of reference genes, the geNorm algorithm was used. MiR-26a and let-7a were identified as the most stably expressed miRNAs, whereas U6 showed the most variable expression levels. Future investigations are planned to identify miRNA in cumulus cells that can be used as markers for oocyte developmental competence. Using a single oocyte-embryo culture system will enable us to retrospectively relate cumulus miRNA expression to the developmental capacity of the oocyte.This work was supported by EU FP7 EpiHealthNet (N°317146).

172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

2014 ◽  
Vol 26 (1) ◽  
pp. 200 ◽  
Author(s):  
C. de Frutos ◽  
R. Vicente-Perez ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Mixed Logit ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

206 EFFECT OF ADDITION OF FOLLICULAR FLUID OR GROWTH DIFFERENTIATION FACTOR-9 ON IN VITRO MATURATION OF PORCINE OOCYTES DENUDED 20h AFTER THE START OF IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 234
Author(s):  
P. Ferré ◽  
T. T. M. Bui ◽  
M. T. Tran ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Dapi Staining ◽  
Experimental Conditions ◽  
Nuclear Maturation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9

The interruption of communication between oocyte and cumulus cells (CC) can trigger meiotic resumption and exogenous additives, such as follicular fluid (FF) and growth differentiation factor-9 (GDF9), can improve oocyte quality and the developmental competence. This study was undertaken to examine if the absence and presence of FF from medium follicles (MF; 3–6 mm in diameter) or recombinant human GDF9 (Biovision, Milpitas, CA, USA) during the first or/and second half of in vitro maturation (IVM) had any effects on IVM of oocytes from small follicles (SF; 0.5–2 mm in diameter) or MF when the oocytes were denuded at 20 h after the start of IVM. Cumulus-oocyte complexes (COC) were aspirated from SF or MF of slaughtered prepubertal gilt ovaries. Groups of ~30 COC were cultured in a 300-μL drop of porcine oocyte medium containing 50 µM β-mercaptoethanol (mPOM) with or without 10% (v/v) FF and/or 100 ng mL–1 GDF9 at 39°C and 5% CO2 in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl c-AMP, 10 IU mL–1 eCG and 10 IU mL–1 hCG. After the first period of IVM, the CC surrounding the oocytes were removed and the denuded oocytes continued culture for IVM with or without FF or/and GDF9 in the absence of dibutyryl c-AMP and gonadotropins in the same medium for another 24 h. At the end of IVM, meiotic progression of the oocytes was examined by DAPI staining. Statistical analyses from at least 4 replicates data were performed by a 2-way ANOVA and a Tukey’s multiple comparisons test. Removal of CC 20 h after the start of IVM significantly improved the incidence of mature oocytes derived from SF (59.2–64.1% v. 41.6–43.1% in controls, P < 0.05) but not from MF (73.1–78.5% v. 70.6–71.8% in controls), whereas regardless of supplementation with FF or GDF9, the maturation rates were always significantly higher in the denuded oocytes from MF (72.4–83.6%) than SF (57.8–66.2%; P < 0.05). Despite of the origin of COC (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM were not affected by supplementation with FF or GDF9 during the first and/or second half of IVM (P > 0.05). In summary, CC removal from COC 20 h after the start of IVM promotes nuclear maturation of oocytes from SF. Exogenous additives such as GDF9 and follicular fluid from MF do not seem to affect the promotion of nuclear maturation in our experimental conditions.

scholarly journals Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhenwei Jia ◽  
Xueli Wang
Keyword(s):  
Natriuretic Peptide ◽  
Basal Medium ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
The Novel ◽  
Bovine Oocyte ◽  
Meiotic Arrest ◽  
Follicle Size

Abstract The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3–8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3–8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

Treatment with cyclic adenosine monophosphate modulators prior to in vitro maturation alters the lipid composition and transcript profile of bovine cumulus–oocyte complexes and blastocysts

2018 ◽  
Vol 30 (10) ◽  
pp. 1314 ◽  
Author(s):  
Eduardo M. Razza ◽  
Mateus J. Sudano ◽  
Patricia K. Fontes ◽  
Fernanda F. Franchi ◽  
Katia Roberta A. Belaz ◽  
...  
Keyword(s):  
Lipid Content ◽  
Lipid Composition ◽  
Transcriptional Profiling ◽  
Ovarian Follicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Membrane Lipid ◽  
Unsaturated Lipids

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus–oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2 h and 16 transcripts after 24 h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Lalantha R. Abeydeera ◽  
Wei-Hua Wang ◽  
Thomas C. Cantley ◽  
Randall S. Prather ◽  
Billy N. Day
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Thiol Compound ◽  
Nuclear Maturation ◽  
Mean Differences ◽  
Pronuclear Formation

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, β-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 μM BME, 0.5 μg/ml LH, 0.5 μg/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20–22 h and then without hormonal supplements for an additional 20–22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5–6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78–89%). The mean differences in penetration rate (69–77%), polyspermy rate (31–40%), male pronuclear formation rate (93–96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32–39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13–15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.

scholarly journals Expression of integrins and GDF9 mRNAs is associated with ovarian follicle size and donor puberty status in pigs

2016 ◽  
Vol 72 (12) ◽  
pp. 750-754
Author(s):  
Paweł Antosik ◽  
Michal Jeseta ◽  
Wiesława Kranc ◽  
Adrian Chachuła ◽  
Artur Bryja ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Growth Differentiation Factor 9 ◽  
Follicle Size ◽  
Ovarian Folliculogenesis ◽  
Subordinate Role ◽  
Lower Expression ◽  
Main Factor

Ovarian folliculogenesis and oogenesis has a significant impact on embryo growth and development in preimplantation stages. Although both processes are widely understood in several species of mammals, including pigs, the factors influencing the proper maturation capability of oocytes, as well as the developmental competence of the surrounding somatic granulosa cells (GCs) and cumulus cells (CCs), are still not entirely known. This study aimed to investigate the expression of growth differentiation factor 9 (GDF9) and integrins (ITGB1, ITGB2, ITGB3 and ITGB4) in porcine oocytes isolated from follicles of various size and donors characterized by different puberty status. The relative abundance of GDF9, ITGB1, ITGB2, ITGB3, and ITGB4 mRNAs in porcine oocytes isolated from medium follicles of cycling sows (MFCS), small follicles of juvenile gilts (SFJG), and small follicles of cycling sows (SFCS) was assessed by an RT-qPCR assay. We found an increased expression of GDF9 in oocytes isolated from the small follicles of juvenile gilts as compared to the other two groups (P<0.001). A significant down-regulation of ITGB1 and ITGB2 oocyte mRNAs collected from medium follicles of cycling sows was observed (P<0.05 and P<0.001, respectively). The ITGB3 mRNA expression was significantly decreased in oocytes isolated from small follicles of juvenile gilts (P<0.001), whereas a lower expression of ITGB4 in oocytes from both medium follicles (cycling sows) and small follicles (juvenile gilts) was observed. In conclusion, GDF9 may be recognized as the main factor regulating follicle growth at early stages of folliculogenesis. The expression of ITGBs is significantly regulated by the puberty status of donor pigs, and different follicular sizes may play a subordinate role in integrin expression during in vivo follicle development in pigs.

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

Sign in / Sign up

Export Citation Format

Share Document