the meiotic cell cycle in mammalian oocytes proceeds up to the diplotene or dictiate 
 stage of the first prophase and stay at this phase before lh surge ( hubbard & terranova , 1982 ; dutta et al . , 2016 ) .
during growth phase of the 
 follicle , the oocyte remains the arrested condition at prophase i. at this time it 
 is incompetent to complete meiosis .
the germinal vesicle intact oocytes in 
 cumulus - oocyte - complex removed from mature antral follicles spontaneously progress 
 to the metaphase ii stage by culture without gonadotropin ( pincus & enzmann , 1935 ; erickson & sorrenson , 1974 ) . on the other hand , the oocytes enclosed 
 in an antral follicle , do not enter meiosis even though these follicles are removed 
 from their environment . by the adding of gonadotropin or releasing from follicle
therefore , some low molecular weight substances in the 
 follicular fluid have been suggested as a mediator for meiotic arrest ( eppig et al .
cyclic adenosine monophosphate ( camp ) is a key signaling mediator in oocyte for 
 meiotic prophase arrest in most species studied .
elevated camp concentration in the 
 oocyte has been demonstrated to maintain meiotic arrest and a transient decrease in 
 camp levels in the oocytes is thought to be a primary trigger for resumption of 
 meiosis .
camp works through protein kinase a ( pka)-mediated phosphorylation of 
 maturation promoting factor ( jones , 2008 ) . 

the levels of camp can be modulated by the activity of camp phosphodiesterase , pde3a 
 in the oocyte ( hambleton et al . , 2005 ) .
on the 
 other hand , camp can be transported from the surrounding cumulus cells ( norris et al . , 2009 ) or generated by oocyte 
 itself through the activation of heterotrimeric g protein ( gs)-linked 
 receptor , gpr3 or grp12 g ( mehlmann , 2005 ) .   in the case of cyclic gmp
, it has been shown to increase in the whole ovaries in 
 hamsters until the lh surge when camp rises and cgmp falls ( hubbard , 1980 ) .
cgmp works in maintaining of the meiotic arrest 
 in oocyte and that this inhibition is dose and time dependent and expressed only in 
 oocytes with intact cumulus ( jankowski et al . , 
 1997 ; zhang et al . , 2010
; robinson et al . , 2012 ; egbert et al . , 2014 ; shuhaibar 
 et al . ,
it appears that the inhibitory action of cgmp on oocyte 
 maturation is mediated by the cumulus oophorous while camp acts directly on the 
 oocyte ( hubbard & terranova , 1982 ) . 

however , so far , the essentiality of cumulus cells for cgmp in oocyte is 
 controversy .
therefore , in the present study , we investigate whether the 
 cgmp - mediated meiotic arrest can be possible without cumulus cells during in 
 vitro culture .
cd1 mice were maintained on a 10 hr light and 14 hr dark cycle under standard 
 vivarium condition and were supplied with food and water ad 
 libitum .
immature female mice ( 20 - 24 days old ) were injected with 5 
 iu of pregnant mares serum gonadotropin ( pmsg ; i.p . ) to enhance multiple 
 follicular development .
oocytes were collected by ovarian follicular puncture with a needle 
 under a dissecting microscope .
to get cumulus free gv oocyte , the cumulus mass 
 were removed with a fine - bore pipetted .
the entire procedure did within 20 min 
 after puncture and only the healthy oocytes and cumulus - enclosed oocytes were 
 chosen for examination .
cumulus - oocyte complexes and oocytes were transferred 
 into bww medium containing 0.4% bsa and cultured .
oocytes ( 8 - 10 oocytes / drop ) were cultured in 10 l drops of bww in mineral oil 
 ( sigma ) or 15 hr at 37 in a humidified atmosphere conditioning 5% 
 co2 .
the progression of meiotic maturation was scored by 
 observing the breakdown of germinal vesicles ( gvb ) and the subsequent appearance 
 of polar bodies with an inverted microscope .
cell permeable cgmp analogs , 
 8-bromoguanosine 3,5-cyclic monophosphate ( 8-br - cgmp ; 1 mm or 1 m ) , 
 rp-8-bromo - cgmps ( 1 mm or 1 m ) , rp - pcpt-8-bromo - cgmp ( 1 mm or 1 m ) were treated 
 into the medium and gv intact oocytes were cultured .
the progression of meiotic 
 maturation was scored by observing the breakdown of germinal vesicles ( gvb ) and 
 the subsequent appearance of polar bodies with an inverted microscope .
chi - squared test was used in statistical analysis , and a p value 
 less than 0.05 was considered significant .
to know the difference of cgmp sensitivity to meiotic arrest by the existence of 
 cumulus , cell permeable cgmp analog , 8-bromoguanosine 3,5-cyclic monophosphate 
 ( 8-br - cgmp ) was treated to gv intact oocytes as mentioned in materials and 
 methods .
gvb was inhibited concentration dependently by the 8-br - cgmp in the 
 cumulus - enclosed oocyte ( fig .
, it also inhibited gvb in denuded oocytes with concentration dependent 
 manner ( fig .
at 6 hr of incubation , 
 the gv intact percentage of cumulus - enclosed oocytes was 0% at control , 21% at 1 
 mm 8-br - cgmp , and 41% at 1 m 8-br - cgmp .
on the other hand , in the case of denuded 
 oocyte , the gv intact percentage was 0% at control , 1.4% at 1 mm 8-br - cgmp , and 
 95% at 1 m 8-br - cgmp . at 15 hr of incubation , the intact percentage was 0% at 
 control , 0% at 1 mm 8-br - cgmp , and 11% at 1 m 8-br - cgmp . in the case of
denuded 
 oocyte , gv intact percentage was 0% both at control and 1 mm 8-br - cgmp , and 85% 
 at 1 m 8-br - cgmp .  
the inhibitory effect of 8-br - cgmp was more severe in denuded oocytes than 
 cumulus - enclosed oocytes . at 1 m 8-br - cgmp ,
the gvb inhibition was significantly 
 higher in denuded oocytes than cumulus - enclosed oocytes at all time points 
 ( fig .
gv oocytes were cultivated in medium containing 0.001 mm or 1 mm 
 8-br - cgmps for 15 hr and nuclear status were determined under the 
 investigated microscope ( nomarski dic modulated ) at 0 hr , 1.5 hr , 3 hr , 
 6 hr , 9 hr and 15 hr .
the cell permeable cgmp analog rp-8-br - cgmp and rp - pcpt-8-br - cgmp but have a 
 different function from 8-br - cgmp were treated to the gv intact oocytes . 

rp-8-br - cgmp and rp - pcpt-8-br - cgmp have antagonistic effects in cgmp target 
 cellular mediator , a cgmp - dependent protein kinase . in cumulus - enclosed oocyte
2a ) . 
 in denuded oocyte , it also did not block the gvb ( fig .
at 6 hr of culture , gv intact percentages in cumulus - enclosed oocytes 
 were 0% both at control and 1 m rp-8-br - cgmp , and 48% at 1 mm rp-8-br - cgmp . in 
 denuded oocytes ,
the gv intact percentages were 4% at control , 70% at 1 mm 
 rp-8-br - cgmp , and 20% at 1 m rp-8-br - cgmp . at 15 hr after culture , the gv arrest 
 percentages in cumulus - enclosed oocytes were 0% both at control and 1 m 
 rp-8-br - cgmp , and 10% at 1 mm rp-8-br - cgmp .
in denuded oocytes , the gv arrest 
 percentages were 0% at control , 10 % at 1 mm rp-8-br - cgmp , and 2.5% at 1 m 
 rp-8-br - cgmp ( fig .
2 ) . compared with the 
 results of 8-br - cgmp in denuded oocytes ,
gv oocytes were treated with 0.001 mm or 1 mm rp-8-br - cgmp combined with 
 750 m guanosine for 15 hr and nuclear status were determined under the 
 investigated microscope ( nomarski dic modulated ) at 0 hr , 1.5 hr , 3 hr , 
 6 hr , 9 hr and 15 hr .
* : 
 p<0.05 versus control . in the case of rp - pcpt-8-br - cgmp ,
its effect on gv arrest was stronger than 
 rp-8-br - cgmp ( fig . 2 and 3 ) .
in cumulus - enclosed oocytes which were 
 cultured in rp - pcpt-8-br - cgmp containing media , the gv arrest percenttages were 
 0% at control , 43% at 1 mm rp - pcpt-8- br - cgmp , and 9.5% at 1 m rp - pcpt-8-br - cgmp 
 after 6 hr of culture ( fig .
the gv arrest percentages were 0% both control and 1 m 
 rp - pcpt-8-br - cgmp , and 23% at 1 mm rp - pcpt-8-br - cgmp .
in cumulus - enclosed 
 oocytes , the gv arrest effect is similar between rp-8-br - cgmp and 
 rp - pcpt-8-br - cgmp . in denuded oocyte ,
the gv arrest percentages after 6 hr of 
 culture were 40% at control and 73% both in 1 mm and 1 m rp - pcpt-8-br - cgmp . at 
 15 hr of culture ,
the gv arrest percentages were 0% at control , 57% at 1 mm 
 rp - pcpt-8-br - cgmp , and 60% at 1 m rp - pcpt-8-br - cgmp . when compared with the 
 results of cumulus - enclosed oocytes and denuded oocytes , the inhibitory effects 
 of rp - pcpt-8-br - cgmp severe in denuded oocytes ( fig .
gv oocytes were treated with 0.001 mm or 1 mm rp - pcpt-8-br - cgmp combined 
 with 750 m guanosine for 15 hr and nuclear status were determined under 
 the investigated microscope ( nomarski dic modulated ) at 0 hr , 1.5 hr , 3 
 hr , 6 hr , 9 hr and 15 hr .
gv ; germinal vesicle , cont ; control . * : 
 p<0.05 versus control .
fertility of female is depending on the quantity and quality of follicles , and the 
 getting a competence for development in the oocyte is critical for oncogenesis . 

oocytes have to stay until ovulation at gv intact status , prophase i of meiosis .
cotreatment of fsh and adenosine 
 markedly inhibits the gvb of ceo in a dose - dependent manner ( mille & behrman , 
 1986 ) .
recently , we reported about the possible roles of adenosine and adenosine 
 receptor - mediated calcium signaling in gv arrest of oocytes enclosed with cumulus 
 ( hwang & cheon , 2013 ) .
it supports the 
 role of calcium in camp action ( powers & paleos , 
 1982 ; preston et al . , 1987 ) .
on 
 the other hand , cyclic gmp also is also suggested as a mediator for camp regulation 
 in oocyte .
cgmp inhibit camp hydrolysis through pde3a ( norris et al . , 2009 ; vaccari et 
 al . , 2009 ) .  
the cgmp levels in follicle are uniformly high ( about 2 - 4 m ) .
the level of cgmp in oocyte is also decreased 
 uniformly to about 100 nm and this decrease causes of meiotic restart ( shuhaibar et al . , 2015 ) .
in follicle cgmp 
 producing guanylyl cyclase natriuretic peptide receptor 2 ( npr2 , guanylyl cyclase b ) 
 is present in all of the granulosa cells , but not in the oocyte ( jankowski et al .
, 1997 ; zhang et al . , 2010 ; robinson et 
 al . , 2012 ; egbert et al . ,
the cell permeable 
 cgmp analog , 8-br - cgmp inhibits gvb with concentration - dependent manners both in 
 cumulus - enclosed oocytes and denuded oocyte .
the effects of 8-br - cgmp were stronger 
 in cumulus - enclosed oocytes than denuded oocytes .
such a responsibility of oocyte to 
 cgmp suggest that cumulus may modulate the cgmp effects on gvb arrest .
the antagonists of cgmp in cellular signaling , rp-8-br - cgmp and rp - pcpt-8-br - cgmp 
 were showed opposite effect to 8-br - cgmp . however , its effects were specific to the 
 concentration of rp-8-br - cgmp in cumulus - enclosed oocytes but not in denuded 
 oocytes . in denuded oocytes ,
the spontaneous maturation processed dependently to the 
 concentration of rp-8-br - cgmp . in cumulus - enclosed oocytes
, the effects of 
 rp - pcpt-8-br - cgmp also had a similar pattern with the rp-8-br - cgmp concentration . in 
 denuded oocytes , rp - pcpt-8-br - cgmp
was significantly inhibited gvb in all groups 
 compared with control , though its gvb inhibitor effect were less than 8-br - cgmp . the 
 responsibility to the rp - pcpt-8-br - cgmpthe ministry of education , science and 
 technology and rp-8-br - cgmp
however , interestingly rp - pcpt-8-br - cgmp inhibited severely 
 more than rp-8-br - cgmp at 1 mm but not 1 m. such differences between antagonists for 
 cgmp may be originated from the difference of the affinity to the target molecule . 
 based on them , it means that the sensitivity for gv arrest to cgmp may be modified 
 by cumulus cells .  
the cgmp transported from granulosa cells to oocyte through gap junction has been 
 suggested as a key factor for gv arrest .
based on the results , we know that the 
 responsibility to agonist or antagonist is modified by the cumulus .
the gv arrest 
 was maintained in denuded oocyte by 8-br - cgmp significantly compared with 
 rp-8-br - cgmp or rp - pcpt-8-br - cgmp
. put together , it suggests that the role of cgmp 
 for spontaneous gvb is modulated by cumulus .
 abstractintact germinal vesicle ( gv ) arrest and release are essential for maintaining the 
 fertility of mammals inducing human . 
 intact germinal vesicle release , maturation 
 of oocytes is maintained by very complex procedures along with folliculogenesis 
 and is a critical step for embryonic development . 
 cyclic guanosine monophosphate 
 ( cgmp ) has been suggested a key factor for meiotic arrest but so far its 
 mechanisms are controversy . in this study 
 we examine the effects of cgmp on 
 germinal vesicle breakdown in cumulus - enclosed oocytes and denuded oocytes . 
 
 spontaneous maturation was inhibited by a cgmp agonist , 8-br - cgmp with 
 concentration dependent manners both in cumulus - enclosed oocytes and denuded 
 oocytes . 
 the inhibitory effect was more severe in denuded oocytes than 
 cumulus - enclosed oocytes . 
 the rp-8-br - cgmp and rp - pcpt-8-br - cgmp did not 
 severely block gvb compared to 8-br - cgmp . 
 the spontaneous gvb inhibitory effects 
 were different by the existence of cumulus . based on them it 
 is suggested that 
 the cumulus modulates the role of cgmp in gv arrest . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION