Meiotic Resumption In Bovine Oocytes Research Articles

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption

BackgroundStudies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. ObjectiveWe aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. Experimentaldesign: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. ResultsqRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). ConclusionN-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes.

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p<0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7h of in vitro maturation (IVM) (p<0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p<0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

350 EFFECT OF INDIRECT MAINTENANCE OF INTRACELLULAR cAMP IN CUMULUS - OOCYTE COMPLEXES USING 3-ISOBUTYL-1-METHYLXANTHINE ON GAP JUNCTIONAL COMMUNICATIONS AND OOCYTE MEIOTIC MATURATION

Oocyte and cumulus cells communicate through an extensive network of gap junctions (GJs), which permit the transfer of small molecules such as cAMP. Gonadotropin strongly enhances the intracellular cAMP concentration in cumulus cells, and induces oocyte meiotic resumption. Enhanced cAMP also triggers a reduction of GJ communications (GJCs) in cumulus-oocyte complexes (COCs), accompanied by cumulus expansion. Intracellular cAMP is modulated by both adenylate cyclase (AC) for synthesizing and phosphodiesterase (PDE) for degrading. Addition of AC to gonadotropin-free medium induces meiotic resumption of bovine oocytes without cumulus expansion, suggesting that maintenance of cAMP at a certain level in COCs may be crucial for either prolonged maintenance of GJCs or the timing of oocyte meiotic resumption. In the present study, we investigated the intracellular cAMP concentrations in porcine COCs or oocytes, and GJCs during in vitro oocyte maturation culture using PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Porcine COCs obtained from prepubertal gilts were cultured for 20 h (1st culture) using M199 containing 10% FCS (basic medium, BM group) with FSH (FSH group) or IBMX (IBMX group). Following this, the COCs were transferred into the basic medium containing FSH and LH, and cultured for another 24 h (2nd culture). At 6, 12, and 20 h of the 1st culture, intracellular cAMP in COCs or oocytes was measured. To determine GJCs in each COC, Lucifer Yellow fluorescent dye was microinjected into cumulus-enclosed oocytes at 6 or 12 h of the 1st culture, and the ability of dye transfer, which is related to the GJCs, from the oocyte to the surrounding cumulus cells was observed. At the end of the 1st culture, 30.8 � 6.0% of the oocytes in the FSH group underwent germinal vesicle breakdown (GVBD), whereas only a few oocytes in the BM group (8.6 � 2.4%) and the IBMX group (5.8 � 3.0%) achieved GVBD (P &amp;lt; 0.05). In contrast, ratios of metaphase-II (M-II) stage oocytes at the end of the 2nd culture did not differ between the FSH group (75.7 � 3.9%) and the IBMX group (68.2 � 6.8%), although a few oocytes in BM group (10.1 � 3.7%) reached the M-II stage (P &amp;lt; 0.05). Concentrations of cAMP in COCs and oocytes increased drastically in the FSH group compared to those of the BM and IBMX groups (P &amp;lt; 0.05). In addition, the concentration of cAMP in IBMX group oocytes was also higher than that in the BM group, with a significant difference detected at 20 h (P &amp;lt; 0.05). The GJCs in the FSH group were gradually closed, depending on the length of time in culture (54.9 � 3.7% of COCs closed their GCJs at 12 h of the 1st culture). In contrast, in the IBMX group, only 23.0 � 3.7% of COCs closed their GJCs at 12 h of the 1st culture, which was significantly different from that of the other two groups (P &amp;lt; 0.05). These results suggest that treatment with IBMX during the first half of IVM culture can induce subsequent meiotic resumption of porcine oocytes, and that a moderate increase of cAMP concentration in COCs or oocytes prolongs GJCs during the treatment.

Effect of type 3 and type 4 phosphodiesterase inhibitors on the maintenance of bovine oocytes in meiotic arrest.

The use of broad-spectrum inhibitors first suggested that phosphodiesterases (PDEs) are involved in the maturation of bovine oocytes. Modulation of individual PDE families is now possible with the use of newly developed type-specific PDE inhibitors. This study evaluated the role of type 3- and type 4-specific PDE inhibitors on the meiotic arrest of bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). It also evaluated the role of these specific inhibitors on meiotic arrest when COCs are incubated in the presence or absence of theca cell monolayers. Bovine COCs were aspirated from ovaries collected at the abattoir. Denuded oocytes and COCs were incubated for 12 h in culture medium alone or culture medium containing the type 3 PDE inhibitors cilostamide (10 and 20 microM) or milrinone (10 and 50 microM) or the type 4 PDE inhibitor rolipram (10 and 50 microM). Oocytes were then fixed and classified according to the status of nuclear maturation. Cumulus-oocyte complexes were coincubated with untreated theca cell monolayers or theca cell monolayers treated with the different specific PDE inhibitors. Bovine COCs or DOs incubated in culture medium resumed meiosis, but supplementation of the culture medium with the PDE3 inhibitors cilostamide or milrinone resulted in meiotic arrest. On the other hand, supplementation of the culture medium with rolipram did not prevent oocyte maturation. Furthermore, PDE3 inhibitors, but not PDE 4 inhibitors, had an additive effect on the inhibitory action of theca cell monolayers on oocyte maturation. These data support the hypothesis that inhibition of PDE3 prevents the meiotic resumption of bovine oocytes, whereas inhibition of PDE4 does not block oocyte maturation even under normally inhibitory conditions. The additive effect of PDE3 inhibitors on the ability of theca cells to maintain bovine oocytes in meiotic arrest suggests that type 3 PDE has an important role in meiotic resumption of bovine oocytes.

Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I

In this study we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, inhibits meiotic resumption in bovine oocytes by blocking germinal vesicle breakdown in a dose-dependent manner. A concentration 100 microM blocked over 60% of oocytes, while 150 microM inhibited almost all oocytes compared to the control in which over 80% resumed meiosis. Following a second 24 hr culture under conditions permissive to normal maturation, almost all (95%) of blocked oocytes resumed meiosis and progressed to metaphase II. In terms of developmental competence, oocytes maintained in meiotic arrest for 24 hr with 100 microM exhibited a similar capacity to develop to the blastocyst stage as nonblocked control oocytes following maturation, fertilization, and culture in vitro. Cryopreservation was employed as a tool to detect differences in the oocyte viability between blocked and control oocytes. Cleavage of oocytes was significantly reduced following vitrification and activation both in BL-I treated (40.2% vs. 71.9%, P < 0.05) and the control groups (45.6% vs. 81.7%, P < 0.05). However, BL-I treated oocytes were less likely to develop into blastocysts following vitrification (20.0% from vitrified vs 42.5% from nonvitrified cleaved oocytes, P < 0.05, based on cleaved oocytes) compared to nontreated oocytes (34.0% from vitrified vs. 42. 9% from nonvitrified oocytes, P < 0.05). These results demonstrate the feasibility of maintaining bovine oocytes in artificial meiotic arrest without compromising their subsequent developmental competence and may represent a tool for improving the development of less competent oocytes.

Role of Calcium in the Resumption of Meiosis I in Bovine Oocytes

Liu, L., Zhang, H., Qian, J. and Fujihara, N. 1999. Role of calcium in the resumption of meiosis I in bovine oocytes. J. Appl. Anim. Res., 15: 97–104. Ovaries obtained from a local slaughterhouse were brought to the laboratory in saline at 30–32C within 3 h. The cumulus-oocyte complexes were aspirated from follicles of 2- 7 mm in diameter with Ca++- and Mg**- free PBS, and then were washed thrice and cultured in TCM 199 buffered with 25μmol/l Hepes containing different Ca** modifying reagents for 9h. Following fixation and staining, the oocytes were observed to determine the stage of meiosis. lOμmol/l Ca++ chelator EGTA inhibited the occurrence of GVBD significantly (p<0.01). When plasma membrane calcium channel was blocked by more than lOμmol/l verapamil, significantly more oocytes with intact GV were obtained than those in control (p<0.05). More than 62.7% of the oocytes cultured in the medium containing EGTA and verapamil were unable to undergo GVBD. Inactivation of Ca++- calmodulin complex by TFP at concentration of lmol/l or higher inhibits significantly the occurrence of GVBD than that of control (p<0.01). When intracellular Ca++ release was prevented by lOOμmol/l TMB-8 or endoplasmic reticulum Ca++- ATPase activation was inhibited by lOOμmol/l thapsigargin, the oocytes were suppressed to proceed GVBD significantly than those in control (p<0.05 or p<0.01). The occurrence of GVBD in the oocytes was influenced significantly by 50μmol/l or more neomycin (p<0.05). The results indicate that both extracellular and intracellular calcium are involved in the regulation of meiotic maturation and intracellular calcium release induced by IP3 may not be the only pathway for meiotic resumption in bovine oocytes.

Co-culture of Cumulus-Enclosed Bovine Oocytes with Theca Cells Induces the Meiotic Arrest but does not Inhibit Germinal Vesicle Development.

The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.

Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes

The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34 cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OAcounteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH-stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes.

Role of the cyclic adenosine monophosphate-dependent protein kinase in the control of meiotic resumption in bovine oocytes cultured with thecal cell monolayers.

It has long been known that intracellular levels of cAMP are implicated in the process of meiotic resumption in bovine oocytes. The present study was undertaken to evaluate the effects of various modulators of cAMP in a coculture system with thecal cell monolayers that have been shown to promote meiotic arrest (germinal vesicle [GV] stage) in bovine oocytes. Ovaries were obtained from a slaughterhouse, and oocytes were collected by puncture of 1- to 5-mm follicles. Cumulus-oocyte complexes (COC) were cultured for 18 h in tissue culture medium 199 supplemented with 10% fetal calf serum and various modulators. When H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinole-sulfonamide) , a protein kinase A inhibitor, was used alone, a significant increase (p < 0.05) in the percentage of COC maintained in GV stage was observed. In the coculture system with thecal cell monolayers that maintained oocytes at the GV stage, H-89 significantly (p < 0.05) decreased this percentage. When oocytes were denuded of their cumulus cells, H-89 (50 microM) alone did not significantly increase the percentage of oocytes maintained at the GV stage. In addition, denuded oocytes were not significantly maintained at the GV stage when cocultured with thecal cell monolayers. In the absence of thecal cell monolayers, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (Bx), and an activator of adenylate cyclase, forskolin (Fk), significantly increased (p < 0.05) the percentage of denuded oocytes maintained at the GV stage. In contrast, Bx-Fk were not effective in maintaining COC at the GV stage. The inhibitory effect of H-89 on COC was significantly increased (p < 0.05) by Bx-Fk. Using thecal cell monolayers with Bx-Fk, the percentage of COC maintained at the GV stage was not significantly decreased. These results suggest that oocytes were maintained in meiotic arrest by inhibition of cAMP-dependent protein kinase by H-89 in cumulus cells. It seems that inhibitory factors originating from thecal cells are regulated by a cAMP-dependent protein kinase. In turn, inhibitory factors released from thecal cells may regulate a cAMP-dependent protein kinase in cumulus cells.

Activation of p34 cdc2 kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34 cdc2 kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34 cdc2 kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34 cdc2 kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34 cdc2. When these oocytes were cultured for 24 h in a medium supplemented with 100 μg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34 cdc2 kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34 cdc2 kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 ± 18 fmol/ h/ oocyte) as compared to that in oocytes cultured for 0 h (46 ± 11 fmol/ h/ oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34 cdc2 kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.

Influence of Follicular Wall on Meiotic Resumption of Bovine Oocytes When Cultured Inside or Outside Hemi-Sections.

We evaluated the importance of the attachment of the oocyte to the granulosa layer, using co-culture of follicular hemi-sections with groups of 10 COCs (cumulus-oocyte complexes) or 10 DOs (denuded oocytes) and oocytes cultured within their own half-follicle. Various prod-ucts acting on protein phosphorylation (IBMX, PMA, H-7) and cellular differentiation (estrous serum, FSH, LH or estradiol) involved in follicular and nuclear maturation were also used to evaluate their effect on the capacity of the follicular wall to inhibit meiosis resumption. When oocyte were cultured within their own half-follicles, none of the treatments used in this study could significantly reverse the inhibitory action of the follicular wall on the meiotic resumption. When the contact between the oocyte and the hemi-section was ruptured (co-culture of COCs and DOs), oocytes responded differently according to whether they were surrounded or not with their cumulus cells, and inhibition of meiosis was lowered particularly with LH and PMA with COCs. These results show that the communication junctions between the oocyte and the follicle are important for the inhibitory action of the follicle and this contact between both overcome the effect of any treatment applied.

Effects of cycloheximide on chromatin condensations and germinal vesicle breakdown (GVBD) of cumulus-enclosed and denuded oocytes in cattle

To determine if newly synthesized protein is imperative for the resumption of meiosis in bovine follicular oocytes collected from small antral follicles, cumulus-enclosed and denuded oocytes were cultured in TCM-199 both with and without various concentrations of the protein synthesis inhibitor, cycloheximide. After 11 h of culture in inhibitor-free medium, all oocytes had undergone germinal vesicle breakdown (GVBD). However, when concentrations of more than 1.0 μg/ml cycloheximide were added to the medium, the meiotic resumption of bovine oocytes was completely blocked. This inhibitory effect of cycloheximide was fully reversible after removal of the inhibitor from maturation media. Germinal vesicle breakdown following removal of cycloheximide occurred twice as fast as in the control medium. Nevertheless, when oocytes were arrested at the germinal vesicle (GV) stage by cycloheximide, a significantly higher proportion of chromatin condensation (40 to 57%) was observed in denuded oocytes than in cumulus-enclosed oocytes (11 to 22%). Thus the cycloheximide treatment could not prevent the chromatin condensation in only denuded oocytes. We conclude that protein synthesis is a prerequisite for GVBD in bovine follicular oocytes and that cumulus cells are responsible for the complementary regulation of the chromatin condensation at the GV stage, regardless of protein synthesis in the oocytes.

Effect of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes

Abstract Meiotic maturation occurs spontaneously when bovine oocytes are removed from their follicles and cultured in vitro. This natural phenomenon in mammals has greatly facilitated the use of in vitro matured oocytes for fertilization and embryo production. Bovine oocytes from small antral (immature) follicles can be matured in vitro up to the stage where they can be fertilized but their average subsequent developmental capacity is limited. Regardless of progress made to improve culture conditions during oocyte maturation and embryo development during the last few years, only one third of the oocytes selected on morphological criteria result in viable embryos, while the other two thirds, cultured in the same conditions, do not. This indicates some deficiency in these oocytes. It is clear that normal cytoplasmic maturation must occur in vitro to produce a good embryo but is the cytoplasm of all oocytes ready to respond to our maturation conditions? If we suppose that a maturing follicle influences the oocyte's ability to support further development, it is essential to understand this process and simulate this effect in vitro. The first step towards this objective requires a culture system that reproduces the ovarian follicular environment for the oocyte, in which nuclear maturation is prevented. In this study, we summarize the effect of each part of the follicle on meiotic resumption in bovine oocytes. Associations between mural granulosa cells and cumulus cells seem to be essential for prevention of nuclear maturation but the health of the follicle and the follicular fluid content are also important.

Effects of granulosa cell co-culture on in-vitro meiotic resumption of bovine oocytes

This study was undertaken to create an in-vitro model using granulosa cell monolayers to replace the role of the follicle in the maturation of bovine oocytes. Cumulus-oocyte complexes were co-incubated with fresh or 7-day granulosa cell cultures (with new or conditioned medium) or with conditioned medium alone, in the presence or absence of IBMX (isobutylmethylxanthine), adenosine or heparin. Progression to the metaphase-II stage was significantly affected by the co-culture of oocytes with bovine granulosa cell monolayers and to a lesser degree when cultured with supernatant alone (conditioned medium). The oocytes attached rapidly to the monolayer, suggesting that the intimate contact between the granulosa cells and the cumulus-oocyte complexes is an important signal for the maintenance of meiotic arrest. Heparin did not prevent maturation itself, but prevented attachment of cumulus-oocyte complexes to monolayers, thereby reducing their inhibitory effect. Adenosine prevented cumulus expansion and reduced maturation and IBMX was an effective inhibitor only in the presence of additional granulosa cells.