Development of functional LH Receptors on pig cumulus-oocyte complexes cultured in vitro by a novel two-step culture system.

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre-cultured for 72 hr in a medium supplemented with dbcAMP. The pre-cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP-dependent pathway. The dbcAMP-treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH.

Procházka R ，Nemcová L ，Nagyová E ，Scsuková S ，Mlynarcíková A ... -  《-》

Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs.

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.

Ozawa M ，Nagai T ，Somfai T ，Nakai M ，Maedomari N ，Fahrudin M ，Karja NW ，Kaneko H ，Noguchi J ，Ohnuma K ，Yoshimi N ，Miyazaki H ，Kikuchi K ... -  《-》

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes.

Shimada M ，Nishibori M ，Isobe N ，Kawano N ，Terada T ... -  《BIOLOGY OF REPRODUCTION》

Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

van Tol HT ，van Eijk MJ ，Mummery CL ，van den Hurk R ，Bevers MM ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Mitogen-activated protein kinase phosphorylation patterns in pig oocytes and cumulus cells during gonadotrophin-induced resumption of meiosis in vitro.

Ebeling S ，Schuon C ，Meinecke B 《ZYGOTE》