An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes

The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1–2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1–2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3–6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes.

stop gene transcription before being capable of sustaining blastocyst formation 12 . Thus, the NSN-to-SN transition is one of the most important aspects in oocyte cytoplasmic maturation. However, the mechanisms for the NSN-to-SN transition are not clear. Thus, the signaling pathways leading to the NSN-to-SN transition must be studied in order to better understand the epigenetic mechanisms of oocyte gene expression and to select oocytes that are more competent for in vitro maturation. Furthermore, such research will also provide new insights into the molecular regulation of somatic cell reprogramming.
Oocytes must be maintained at the GV stage in order to observe the NSN-to-SN transition in vitro. However, oocytes from many species quickly undergo GVBD after being released from follicles, and thus, GVBD must be inhibited with drugs to observe in vitro the NSN-to-SN transition in these species. Worse yet, drugs used for GVBD inhibition are often non-specific; they may affect the NSN-to-SN transition as well. Thus, oocytes with a naturally extended GV stage in vitro are ideal for studying NSN-to-SN transition. Previous studies indicated that porcine oocytes could maintain GV intact in vitro for an extended period of over 20 h 15 , and those from small follicles showed a high percentage (over 60%) of NSN configuration at the first release from follicles 8 . The aim of the present study was to explore the signaling pathways leading to oocyte NSN-to-SN transition by using pig oocytes from small antral follicles. The results have the first time up-to-date explored the signaling pathways leading to oocyte NSN-to-SN transition and established an essential role for intra-oocyte MAPK in the NSN-to-SN transition. The data not only will contribute to our understanding of the epigenetic mechanisms for oocyte maturation but also will provide important models for research on regulation of DNA transcription and the epigenetics and reprogramming in somatic cells.

Results
Classification of GV chromatin configuration and RNA transcription. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on disappearance of nucleolus and nuclear membrane (Fig. 1). The GV0 configuration was characterized by a distinct nucleolus and a diffuse, filamentous pattern of chromatin in the whole GV area. In GV1, the nucleolus was surrounded by a complete heterochromatin ring and heterochromatin was not obvious in the nucleoplasm. In GV2 and GV3, the heterochromatin ring around the nucleolus was often incomplete or forming a horseshoe, and clumps and strands of heterochromatin were observed in the GV. In GV4, the heterochromatin clumps or strands remained but the nuclear membrane was less distinct and the nucleolus disappeared completely. For convenience, GV0 was designated as NSN configuration, while GV1, GV2 and GV3 were classed as SN configuration in this study. Gene activities in oocytes with different chromatin configurations were determined by observing global RNA transcription after 5-ethynyl uridine (EU) labeling. Whereas the NSN (GV0) oocytes showed an intensive RNA transcription, no transcription was observed in GV1 and GV2 oocytes, and only faint labeling was observed in the GV3 oocytes (Fig. 1). Oocytes freshly collected from 1-2 mm follicles contained too few GV4 oocytes to observe RNA transcription.
Role of MAPK in regulating the NSN-to-SN transition. As MPF and MAPK are well-known molecules regulating GVBD, their roles in modulating NSN-SN transition were observed. Because around 60% of the oocytes from 1-2 mm follicles displayed NSN configurations while all the oocytes from 3-6 mm follicles had a SN configuration, the intra-oocyte MPF and MAPK activities were measured in these oocytes. The MAPK activity was significantly higher in oocytes from 1-2 mm follicles than in oocytes from 3-6 mm follicles ( Fig. 2A). However, the MPF activity was hardly detectable in oocytes from either 1-2 or 3-6 mm follicles although it was obvious in GVBD oocytes (Fig. 2B). The results suggested that MAPK, but not MPF, was involved in regulating the NSN-to-SN transition. To further study the role of MAPK in regulating the NSN-to-SN transition, COCs and DOs from 1-2 mm follicles were cultured with or without MAPK inhibitor, U0126. At 16 h of culture, over 90% of the oocytes showed intact GV with or without U0126. Whereas many NSN COCs had taken on the SN configuration without U0126, almost all the NSN DOs remained at the NSN stage (Fig. 2C). Culture with U0126 significantly facilitated the NSN-to-SN transition in DOs (Fig. 2C) while eliminating their MAPK activities almost completely (Fig. 2E). Furthermore, after culture without U0126, the level of intra-oocyte p-MAPK was significantly lower in COCs than in DOs (Fig. 2E). The results suggested that intra-oocyte MAPK inhibited the NSN-to-SN transition, whereas CCs facilitated the transition. Inactivation of protein kinase A (PKA) is required for activation of MAPK and inhibition of the NSN-to-SN transition. To answer the questions how the intra-oocyte MAPK activities are regulated and how CCs facilitate oocyte NSN-to-SN, effects of PKA activation on the NSN-to-SN transition were observed because PKA works upstream of MPF/MAPK 16 . COCs or DOs were cultured with or without PKA activator, db-cAMP. Although db-cAMP facilitated the NSN-to-SN transition of DOs as did MAPK inhibition, it inhibited NSN-to-SN in COCs (Fig. 2D) contrary to the effect of MAPK inhibition. After culture without db-cAMP, the level of intra-oocyte cAMP in COCs was significantly higher than that in DOs (Fig. 2F). Furthermore, oocytes from 1-2 mm follicles contained significantly less intra-oocyte cAMP than did the oocytes from 3-6 mm follicles (Fig. 2G). The results suggested that a lower level of intra-oocyte cAMP and hence inactivation of PKA activated MAPK, which inhibited the NSN-to-SN transition.
MAPK prevents the NSN-to-SN transition by activating NF-κB (nuclear factor kappa-lightchain-enhancer of activated B cells) while inhibiting histone deacetylases (HDACs). How did the intra-oocyte MAPK prevent NSN-to-SN? In somatic cells, the phosphorylation (activation) of nuclear NF-κ B results in its association with CBP/p300 displacing HDAC-1 from DNA, leading to transcription activation 17 . MAPK can activate NF-κ B through phosphorylation (inactivation) of inhibitory κ Bs (Iκ Bs) 18 . We thus hypothesized that intra-oocyte MAPK might prevent NSN-to-SN by activating NF-κ B. Roles of HDACs and NF-κ B in regulating NSN-to-SN were therefore observed. COCs or DOs were cultured for 16 h with HDACs inhibitor, TSA or NF-κ B inhibitor, PDTC. Whereas TSA inhibited NSN-to-SN in COCs (Fig. 3A), PDTC promoted it in DOs (Fig. 3B), suggesting that while HDACs promoted, NF-κ B inhibited NSN-to-SN. Compared to oocytes from 1-2 mm follicles, oocytes from 3-6 mm follicles contained significantly more intra-oocyte Iκ Bα (Fig. 3C) but less p-MAPK ( Fig. 2A). After culture without inhibitors, the level of intra-oocyte Iκ Bα was higher significantly in cultured COCs than in cultured DOs (Fig. 3D). Furthermore, whereas treating DOs with U0126 dramatically increased the expression of Iκ Bα (Fig. 3D), treating DOs with OA to activate MAPK decreased Iκ Bα significantly (Fig. 3E). Because all the oocytes from 3-6 mm follicles are of SN configuration, we propose that by inactivating Iκ B, intra-oocyte MAPK activates NF-κ B, which prevents NSN-to-SN by displacing HDACs from DNA. Fig. 2 indicated that whereas inhibiting MAPK facilitated, exposure to db-cAMP inhibited NSN-to-SN in COCs although db-cAMP facilitated NSN-to-SN in DOs. This suggested that db-cAMP had made CCs send signals that activated MAPK in the oocyte. In the LH receptor-activated signaling pathways regulating meiotic maturation, elevated levels of cAMP in CCs reduce cyclic guanosine monophosphate (cGMP) delivery to the oocyte and induce GVBD by down regulating NPR2 and blocking GJC via activating MAPK 19 . We thus hypothesized that elevated levels of cAMP in CCs would inhibit NSN-to-SN by reducing cGMP delivery to the oocyte. To test this hypothesis, role of GJC in regulating NSN-to-SN was first observed. DOs were cultured with or without dispersed CCs. No significant difference in the percentage NSN oocytes was observed between DOs cultured alone and DOs co-cultured with CCs ( Fig. 3F). Effects of in situ blocking GJC on NSN-to-SN transition were then examined. COCs were cultured for 16 h with or without GJC blocker carbenoxolone (CBX) before chromatin configuration examination. Culture with CBX significantly inhibited the NSN-to-SN transition in COCs (Fig. 3G). The results confirmed that GJC were essential for CCs to facilitate the NSN-to-SN transition.

Elevated cAMP levels activated MAPK while down regulating NPR2 in CCs.
To further confirm our hypothesis that cAMP in CCs inhibits NSN-to-SN by reducing cGMP delivery to the oocyte, effects of cAMP elevation on the activities of MAPK and NPR2 were observed in CCs. When COCs were cultured, the presence of db-cAMP increased the level of p-MAPK in CCs ( 4D) than did CCs from 3-6 mm follicles. The results suggested that, in CCs of small follicles, elevation of cAMP resulted in more p-MAPK but less NPR2, leading to a decrease in the cGMP delivery into the oocyte.

FSH postponed the NSN-to-SN transition by activating MAPK while down regulating NPR2 in CCs.
In the pig, FSH secretion is relatively constant throughout the estrous cycle 20 and the growth of 1.1 to 2 mm follicles is FSH-dependent 21 . Furthermore, treatment with FSH stimulates cAMP accumulation in rat granulosa cells 22 . We thus proposed that FSH might inhibit NSN-to-SN by activating MAPK while down regulating NPR2 in CCs. We therefore observed the effects of FSH in culture media on NSN-to-SN in oocytes and on levels of p-MAPK, Npr2 mRNA and cAMP in CCs. COCs or CCs were cultured for 14 h with or without FSH supplementation. Percentages of the NSN oocytes were significantly higher in oocytes cultured with than without FSH supplementation (Fig. 5A). Whereas p-MAPK level increased (Fig. 5B), the level of Npr2 mRNA decreased (Fig. 5C) significantly in CCs in the presence of FSH. Although culture of COCs with FSH alone did not increase cAMP in CCs, culture of CCs with FSH alone or culture of COCs with both FSH and oocyte-derived paracrine factor (ODPF) inhibitors (SB431542 for GDF9 and LDN193189 for BMP15) significantly increased the cAMP level in CCs (Fig. 5D,E). Furthermore, follicular fluid from 1-2 mm follicles contained more FSH than did that from 3-6 mm follicles (Fig. 5F). The results (a) confirmed that FSH in culture media and in small follicles postponed NSN-to-SN by activating MAPK while down regulating NPR2 in CCs, and (b) suggested that ODPF inhibited FSH actions on cAMP production.
ODPF and estradiol (E2) facilitated NSN-to-SN by enhancing CCs' cGMP production and delivery to the oocyte. There are reports that ODPF promotes Npr2 expression and elevates cGMP levels in CCs 23 , and that E2 is essential for the promotion and maintenance of NPR2 expression 24 . We thus hypothesize that 3-6 mm follicles would contain more ODPF and E2 in follicular fluid and more NPR2 in CCs than would 1-2 mm follicles, and that culture of COCs with ODPF inhibitors would inhibit NSN-to-SN and culture with E2 would increase NPR2 levels in CCs. Our measurement showed that the levels of BMP-15 and E2 in follicular fluid were significantly higher in 3-6 mm follicles than in 1-2 mm follicles (Fig. 6A,B). Figure 4D shows that CCs from 3-6 mm follicles contain more Npr2 mRNA than did those from 1-2 mm follicles. Furthermore, culture of COCs with ODPF inhibitors significantly inhibited oocyte NSN-to-SN (Fig. 6C) and culture with E2 increased NPR2 levels in CCs (Fig. 6D). The results suggested that ODPF and E2 facilitated NSN-to-SN by enhancing cGMP production and delivery to the oocyte.

Discussion
By manipulating enzyme activities in vitro and by measuring related molecules both in vivo and in vitro, this study has established a pivotal role for MAPK in the regulation of the NSN-to-SN transition. Thus, the present results showed that the intra-oocyte MAPK inhibited GV chromatin condensation (the NSN-to-SN transition) of pig oocytes. Our further observation indicated that within the oocyte in small follicles, a lower level of cAMP activated MAPK, which activated NF-κ B by inactivating Iκ B (Fig. 7). Activated NF-κ B displaces H1 and HDAC from DNA, leading to chromatin decondensation (NSN). The roles of MAPK in follicle genesis/development, particularly those in meiosis progression, have been studied in several laboratories. For example, activated ERK 1/2 MAPK were localized in the cytoplasm of oocytes from gilts and sows, and their intensity did not differ among primordial/primary, secondary and tertiary follicles 25 . Although activation of MAPK in CCs is necessary for gonadotropin-induced GVBD of cultured COCs, MAPK activation is not required for spontaneous GVBD of cultured DOs 26 . Furthermore, by knocking out Erk1 and Erk2 in mouse oocytes, Zhang et al. 27 observed that ERK1/2 activities in oocyte are dispensable for primordial follicle maintenance, activation and follicle growth. However, both the Mos null oocytes 28 and the ERK1/2-deleted oocytes show anomalies during post-GVBD maturation and/or pronuclear formation, leading to subfertility or infertility. Taken together, the above data suggested that (1) for the first time, the current results demonstrated that compared to oocytes from medium-sized antral follicles, porcine oocytes from small follicles contained more activated MAPK, which inhibited the NSN-to-SN transition; (2) although the intra-oocyte MAPK activity is dispensable for GVBD, it plays an essential role in the NSN-to-SN transition; and (3) the abnormalities observed in the Mos null and ERK1/2-deleted mouse oocytes during post-GVBD maturation and pronuclear formation might suggest that due to a lack of inhibiting MAPK activities, a premature NSN-to-SN transition had occurred in these oocytes, which shut down the transcription of factors essential for oocyte final maturation and early embryo development.
It is well-known that MPF/MAPK facilitates post-GVBD chromosome condensation, but their role in GV chromatin condensation and DNA transcription has not been reported. Furthermore, the inhibitory effect of MAPK on GV chromatin condensation (NSN-to-SN transition) seems contradictory to their role in post-GVBD chromosome condensation. Studies on histone acetylation also indicated different mechanisms for the GV chromatin condensation and the post-GVBD chromosome condensation. For example, fully grown mouse oocytes (mostly of SN configuration) were fully acetylated at all the lysine residues on H3 and H4, but underwent deacetylation after GVBD 1,29 . Thus, the different mechanisms between the GV chromatin condensation and the post-GVBD chromosome condensation will be an very interesting topic in future studies.
It is known that NF-κ B activation leads to expression of target genes by regulating chromatin structure 30 , and that NF-κ B is maintained in a latent form in the cytoplasm by means of sequestration by Iκ B proteins. A significant increase of the Iκ Bα -protein was observed as the NF-κ B/p65-binding activity decreased with transcription silencing during the transition from fully-grown immature to in vitro matured MII bovine oocyte 31 . It was shown in somatic cells that MAPK could activate NF-κ B through inactivating Iκ Bs. For example, in human embryonic kidney 293 cells, over-expression of MEKK1 preferentially stimulates the kinase activity of IKKβ , which resulted in inactivation of Iκ Bs 18 . Furthermore, MEKK1 stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro 32 . There are also numerous reports that NF-κ B activation displaces histone H1 and HDAC from DNA, leading to chromatin relaxation. For example, activated nuclear NF-κ B can bind to CBP/p300 and displace HDAC-1 from DNA 17 , and it was able to displace histone H1 and prevented its binding to nucleosome 33 . In addition, the NSN-to-SN transition was significantly impaired in the HDAC2 knockout (Hdac2 −/− ) mice 34 .
The present results indicated that in CCs of the small porcine follicles, a series of molecular events resulted in reduced production and delivery of cGMP into the oocyte, leading to a decline in intra-oocyte cAMP that inhibited the NSN-to-SN transition (Fig. 7). Firstly, the current results suggested that in small follicles, both a lower level of E2 and ODPF and a higher level of FSH inhibited the NSN-to-SN transition. According to Knox 35 , about 54% of the medium-sized (3-7 mm) porcine follicles are from ovaries at the early follicular, pro-estrous and estrous stages when large dominant follicles exist, but only about 44% of the small (< 3 mm) follicles are from ovaries containing large dominant follicles. It is known that the dominant follicles produce a large quantity of estrogen, which turns down the pituitary secretion of FSH 36 . Thus, it was expected that most of the medium (subordinate) follicles would suffer FSH insufficiency and undergo atresia at this stage of follicle selection 37 . In both cows 38 and sows 39 , the concentration of E2 increased significantly with increasing follicular sizes. Studies in the human suggested that GDF9 levels in the follicular fluid were highly correlated with oocyte nuclear maturation and embryo quality 40 . Porcine oocytes from < 2 mm follicles showed significantly lower rates of maturation and blastocyst formation than oocytes from 3-6 mm follicles did after in vitro maturation 41 . Furthermore, a significant positive correlation was observed between BMP-15 and E2 levels in the same human follicle 42 .
Secondly, the present results suggested that in CCs of 1-2 mm follicles, FSH elevated cAMP levels, which activated MAPK while decreasing NPR2 expression (Fig. 7). There are reports that FSH stimulated cAMP accumulation during in vitro culture of rat granulosa cells 22,43 . LH-dependent activation of MAPK has been observed in granulosa cells of different species including the pig 44 . Further observations indicated that the LH-dependent MAPK activation occurred downstream of cAMP and was dependent on PKA activation 45 , and it might involve multiple signaling cascades downstream of the LH receptor, including the EGF receptor and possibly PKC pathways 46 . Likewise, the response of granulosa cells to FSH is also mediated by cAMP/PKA signaling and involves MAPK activation 45,47 . Furthermore, in their summary of the LH-induced EGFR trans-activation pathways leading to oocyte maturation, Conti et al. 19 documented that cAMP inhibits cGMP production through the EGF network. In this study, culture of COCs with EGF postponed the NSN-to-SN transition to the same extent as did culture with FSH (data not shown). However, in the summary by Conti et al. 19 , whether cAMP can suppress NPR2 production directly is in question. Thus, the present results have provided the first direct evidence that cAMP inhibits cGMP production by decreasing NPR2 expression in CCs of small porcine follicles.
Thirdly, the current results suggested that in small porcine follicles, a lower level of ODPF and E2 inhibited the NSN-to-SN transition by reducing NPR2 expression and by removing the ODPF inhibition on FSH actions (Fig. 7). Furthermore, the activated MAPK prevented cGMP delivery into oocytes by closing GJC between CCs and the oocyte. It has been reported that ODPF, particularly the GDF-9-BMP-15 heterodimer, promotes Npr2 expression and elevates cGMP levels in CCs 23 . Whereas in vitro treatment of mouse COCs with FSH stimulated only a transient cAMP rise in CCs and oocytes, which disappeared after a 30 min culture 48,49 , FSH significantly increased cAMP production during a 48-h culture of rat granulosa cells 22,43 . There are reports that ODPF inhibits FSH actions with decreased cAMP production. For example, treatment of rat granulosa cells with GDF-9 43 , BMP-6 50 , or BMP-9 22 significantly suppressed FSH-induced cAMP synthesis. Furthermore, BMP-15 inhibits FSH action by suppressing FSH receptor expression 51 . The ability of natriuretic peptide type C (NPPC) to keep meiotic arrest in cultured mouse COCs and the ability of CCs to produce cGMP were lost in the absence of E2, suggesting that E2 promotes and maintains expression of NPR2 in CCs and participates in the NPPC-mediated maintenance of oocyte meiotic arrest in vitro 24 . Furthermore, MAPK was found to mediate LH-induced oocyte maturation by interrupting GJC within the ovarian follicle through phosphorylation of connexin 43 52,53 .
In summary, the present results suggested that multiple factors including FSH, E2 and ODPF control the oocyte NSN-to-SN transition by acting on CCs, and that CCs regulate the transition by altering intra-oocyte cAMP levels via controlling cGMP production and delivery. Thus, in CCs of 1-2 mm follicles, a lower level of ODPF and E2 reduces NPR2 while enhancing FSH and cAMP actions (Fig. 7). FSH elevates the level of cAMP, which decreases NPR2 while activating MAPK. Activated MAPK closes the GJC, which, together with the NPR2 decrease, reduces cGMP delivery leading to a cAMP decline within the oocyte. Within the oocyte, the cAMP decline activates MAPK via inactivating PKA. By inactivating Iκ B, MAPK activates NF-κ B, which displaces H1 and HDAC from DNA. As a result, oocytes remain at NSN. In 3-6 mm follicles, a significant increase in ODPF and E2 and a FSH shortage initiate a reversion of the above events leading to chromatin condensation (SN) in the oocyte. The results have the first time up-to-date explored the signaling pathways leading to oocyte NSN-to-SN transition in the mammalian species and have established an pivotal role for intra-oocyte MAPK in the regulation of the NSN-to-SN transition and hence DNA transcription. The data not only will contribute to our understanding of the epigenetic mechanisms for oocyte maturation but also will provide important models for research on the epigenetics and reprogramming in somatic cells.
Whereas some of the COCs were cultured directly after collection, others were mechanically denuded of cumulus cells (CCs) before culture as denuded oocytes (DOs). The COCs or DOs were cultured in groups of 20 in microdrops of 100 μl covered with mineral oil, at 38.5 °C under 5% CO 2 in humidified air. To culture CCs, CCs released during oocyte denudation were washed twice in D-PBS by centrifugation (1500 × g, 5 min). Pellets were resuspended, and cells were counted on a hemocytometer. The final suspension (2-5 × 10 5 cells/ml) was added to wells of 96-well culture plates (200 μl/well) and cultured at 38.5 °C in a humidified atmosphere of 5% CO 2 in air.

Observation of GV chromatin configuration and GVBD.
Oocytes were denuded of CCs by pipetting in D-PBS containing 0.1% hyaluronidase. Oocytes were labeled for 10 min in D-PBS containing 10 mg/ml Hoechst 33342 at 38.5 °C under 5% CO 2 in humidified air. Oocytes were then placed on glass slides and compressed with coverslips to visualize GV. The mounted oocytes were observed under a Leica DMLB microscope equipped with a CCD camera. Oocytes were first examined with phase contrast to visualize morphology of nucleoli and nuclear envelope, and then observed with fluorescence optics. Hoechst fluorescence was obtained by excitation at 220-360 nm using a mercury lamp (50 W) attenuated with neutral filters.