Increasing the cAMP concentration during in vitro maturation of pig oocytes improves cumulus maturation and subsequent fertilization in vitro.

Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus-oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX- and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus-oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP- (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP- (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be detected between the blastocyst rate of dbcAMP- and dbcAMP+ (17.1% and 21.0% on Day 7, respectively; P > 0.05). In conclusion, the use of IBMX during collection did not cause a meiotic arrest. Using dbcAMP during IVM caused a greater normal fertilization rate, a lower rate of adherent COCs during IVM, higher levels of ADAMTS-1 in cumulus cells, and an equal blastocyst rate after screening out adherent COCs. These findings contribute to a better understanding of cAMP involvement in porcine oocyte maturation and provide a basis to develop an improved system with less polyspermy and higher blastocyst rates.

Appeltant R ，Beek J ，Vandenberghe L ，Maes D ，Van Soom A ... -  《-》

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence.

Zeng HT ，Ren Z ，Guzman L ，Wang X ，Sutton-McDowall ML ，Ritter LJ ，De Vos M ，Smitz J ，Thompson JG ，Gilchrist RB ... -  《-》

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes.

Somfai T ，Kikuchi K ，Onishi A ，Iwamoto M ，Fuchimoto D ，Papp AB ，Sato E ，Nagai T ... -  《ZYGOTE》

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Li HJ ，Sutton-McDowall ML ，Wang X ，Sugimura S ，Thompson JG ，Gilchrist RB ... -  《-》

Effect of dibutyryl cyclic adenosine monophosphate on reactive oxygen species and glutathione of porcine oocytes, apoptosis of cumulus cells, and embryonic development.

Park SH ，Yu IJ 《-》