A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes

1985 ◽  
Vol 11 (1) ◽  
pp. 83-97 ◽  
Author(s):  
Stephen M. Downs ◽  
John J. Eppig
Keyword(s):  
Follicular Fluid ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Meiotic Arrest ◽  
Fluid Component

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.

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's buffered medium, mTBM) and placed in petri dishes containing 50 µL 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°C. It was then centrifuged at 1200g for 3 min and standardized for 1 × 105 spermatozoa mL−1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5°C and 5% CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 µL), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1% orcein in 45% acetic acid and evaluated under phase-contrast microscopy at a 400× magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 ± 9.6% (162/412), and no difference was observed in comparison with the T2 group, 29.5 ± 4.9% (113/383). The monospermic penetration rate was 31.5 ± 6% (51/162) in the T1 group and differed from that in the T2 group, 71.7 ± 3.3% (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 ± 6% (111/162), compared with the T2 group, 28.3 ± 3.3% (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP + PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

2019 ◽  
Author(s):  
Bella Grigorenko ◽  
Igor Polyakov ◽  
Alexander Nemukhin
Keyword(s):  
Adenylyl Cyclase ◽  
Bond Cleavage ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Md Simulations ◽  
Coordination Shell ◽  
Energy Profile ◽  
One Step ◽  
Type V

<p>We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of Mg<sub>A</sub><sup>2+ </sup>are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of Mg<sub>A</sub><sup>2+ </sup>does not include the O<sub>δ1</sub> atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O<sup>3'</sup>H<sup>3' </sup>group in ATP to Asp440 via a shuttling water molecule and P<sup>A</sup>-O<sup>3A</sup> bond cleavage and O<sup>3'</sup>-P<sup>A</sup> bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, Mg<sub>A</sub><sup>2+</sup> is bound to the O<sub>δ1</sub>(Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O<sup>3'</sup>H<sup>3' </sup>group via shuttling water molecules. </p>

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