Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 58-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the nhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4-23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44h treatment induced cumulus expansion but inhibited oocyte maturation while 2h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4-23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4-23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein (s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein (s).

The present study is to investigate the immunolocalization of endothelial and inducible nitric oxide synthase (eNOS, iNOS) in porcine ovary and the effect of nitric oxide (NO) on antrum formation and oocyte meiotic resumption. In Experiment 1, preantral follicles (250-300m in diameter) were cultured in 0 (Control), 0.1, 0.3, 0.5 or 1mM sodium nitroprusside (SNP), a NO donor. In Experiment 2, the cumulus-oocyte complexes (COCs) aspirated from medium follicles (3-6mm in diameter) were incubated in 0.1mM SNP or two inhibitors for NOS, 10mM aminoguanidine bicarbonate salt (AG) or 1mMN-nitro-l-arginine methyl ester (l-NAME), alone or concomitantly. In Experiment 3, ovarian tissues, corpus luteum (CL), corpus albican (CA) and COCs from small (1-2mmin diameter), medium (3-6mm) and large follicles (7-10mm) were isolated, rinsed, fixed, paraffin embedded and stained by the conventional avidin-biotin complex method for the detection of eNOS and iNOS production. The results showed that 0.1mM SNP had no effect on antrum formation (P>0.05) while 0.3, 0.5 or 1mM significantly inhibited the antrum formation (P<0.05). AG markedly inhibited porcine oocyte meiotic resumption (P<0.05) while l-NAME inhibited first polar body (PB1) extrusion (P<0.05). The immunoreactivity of eNOS in early antral follicles was restricted to oocyte and it increased from small, medium to large follicle-enclosed oocytes. Cumulus cells from large follicles showed weak eNOS immunoreactivity but those from small or medium follicles not. In CL, eNOS-positive staining was shown in granulosa lutein cells. In CA, it was in some parenchymal cells. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle or the COCs aspirated from small and medium follicles. The large follicle-enclosed oocyte showed weak immunoreactivity. In CL, some granulosa lutein cells showed iNOS-positive cytoplasm. Such immunostaining was not found in CA. The results demonstrate that porcine ovaries have distinct cell-specific expression of both eNOS and iNOS, and that NO derived from both NOS is actively involved in meiotic resumption. Nitric oxide is not involved in the antrum formation of preantral follicles but exogenous NO inhibits the antrum formation. Endothelial nitric oxide synthase and inducible nitric oxide synthase might be differently functional in CL development and regression.

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1nM ANP. Treatment with CANP-4-23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 50-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted UNCORRECTED PROOF in a higher proportion (P<0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte (P<0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine saltstored oocytes by frozen-thawed giant panda spermatozoa.

Meiosis-activating sterol (MAS) has been shown to induce mouse oocytes cultured in the presence of hypoxanthine (HX) to resume meiosis. The present research was conducted to determine whether amphotericin B or ketoconazole (a promoter and an inhibitor of production of MAS), affected oocyte maturation. Mouse cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) were cultured for 24h in the presence of 4mM HX with FSH or amphotericin Bor ketoconazole. At the end of the culture, the frequency of germinal vesicle break down (GVBD) and polar body formation (PB) were recorded. The results demonstrated: (i) FSH (10-200 IU: l) induced dose-dependent oocytes maturation in CEO, but was without effect on DO. A maximum increase in GVBD and PB was observed with 25-50 IU: l FSH. The presence of FSH (50 IU: l) for 1h was sufficient to induce meiotic resumption, which after 2h reached a plateau similar to that of a continuous presence of FSH. (ii) CEO exposed to amphotericin B (0.0025-2.5 mg: l) underwent GVBD dose-dependently, whereas no effect was observed on DO. The presence of amphotericin B (0.025mg: l) for 1 h stimulated oocyte resumption in a way similar to that of FSH. (iii) Amphotericin B (0.025mg: l) and FSH (50 IU: l) did not show any additive effect on resumption of meiosis. (iv) Ketoconazole (107-103M) inhibited the effect of FSH on resumption of meiosis, but had no effect on oocyte spontaneous maturation. These results show that FSH and amphotericin B induce resumption of meiosis and indicate that they are likely to cause an accumulation of meiosis activating sterols in the CEO, but ketoconazole blocks the production of MAS. The present study supports the notion that MAS plays a physiological relevant role in triggering resumption of meiosis in mouse oocytes.

It has been reported that protein kinase C (PKC) activation participated in the porcine and bovine oocyte maturation, but not in mouse oocyte maturation in vitro. In the present study, the activators and inhibitors of protein kinase A (PKA) (forskolin, DPKI and MDL-12230A) or PKC (PMA, staurosporine and sphingosine) were used to investigate the in vitro effect of PKA or PKC on spontaneous murine oocyte maturation, oocyte resumption of meiosis from HX inhibiting medium (medium+HX), and follicle stimulating hormone (FSH)-induced oocyte maturation. The results showed that when cumulus cell nclosed oocytes (CEOs) or denuded oocytes (DOs) were cultured for 24h in the medium supplemented with forskolin (5M), an activator of adenylate cyclase, the spontaneous oocyte maturation were inhibited. A transient exposure (2h) to forskolin (2-10M) in the medium+HX, and then transferred to a new medium+HX for the further culture, stimulated CEO resumption of meiosis. CDPKI (10-10-10-6M), an inhibitor of PKA, also stimulated oocyte meiotic maturation of CEO in the medium+HX, but not on DO. However, MDL-12230A (10-12-10-9M), an inhibitor of adenylate cyclase, did not promote oocyte maturation in HX arrested CEO. CDPKI (10-10-10-6M) or MDL-12230A (10-12-10-9M) had no effect on FSH-stimulated oocyte meiotic resumption, except at high doses of CDPKI (10-7-10-6M) or MDL-12230A (10-9 M) which inhibited the FSH-induced formation of the first polar body (PB1). An activator of PKC, PMA (10-11-10-7M) dose-dependently inhibited spontaneous oocyte maturation of CEO or DO. Inhibitors of PKC, staurosporine (10-9-10-6M) or sphingosine (10-8-10-5M) induced oocytes in CEOs to resume meiosis in the presence of HX in a dose dependent manner, but had no effect on DOs. FSH 50IU/L) stimulated mouse oocytes in CEOs to override the arrest of HX and resume meiosis, while PMA, at the level of 10-8-10-6M, dramatically inhibited the stimulatory effect of FSH. These results indicate that PKC or PKA may be implicated in the regulation of mouse oocyte maturation. Thus while sustained high level of cAMP or PKA inhibit the resumption of meiosis, a transient rise in cAMP or PKA levels promotes oocyte maturation. The activation of PKC can also block oocyte meiotic resumption. Thus the inactivation of PKC, instead of the transient rise of PKA activity, appears to be involved in the process of FSH-mediated oocyte meiotic maturation.

This study examined the effect of atrial natriuretic peptide (ANP) on porcine cumulus-enclosed oocyte (CEO) maturation and cumulus expansion. ANP negatively regulated follicle-stimulating hormone (FSH)-stimulated germinal vesicle breakdown (GVBD; 90.1, 81.2 and 68.2% for FSH, FSH+10nM ANP and FSH+1mM ANP, respectively), first polar body emission (PB1; 86.1, 75.3 and 53.3% for FSH, FSH+1nM ANP and FSH+1mM ANP, respectively) and cumulus expansion (CEI; 3.47, 3.16 and 2.43 for FSH, FSH+1nM ANP and FSH+1mM ANP, respectively) in a dosedependent manner when CEOs were cultured in the maturation medium containing porcine follicular fluid (pFF). This negative effect showed a time-dependent manner after preincubation with 100 Nm ANP for 5h (78.4% PB1), 10h (81.7% GVBD and 74.1% PB1), 20h (78.5% GVBD and 68.9% PB1), and 44h (75.3% GVBD and 60.5% PB1), respectively. ANP also significantly inhibited FSHinduced porcine oocyte GVBD (47.6% versus 83.8%) and PB1 emission (22.4% versus 45.2%) when CEOs were cultured in pFF-free maturation medium. cGMP analog 8-Br-cGMP (10mM to 1mM) mimicked the effects of ANP on GVBD, PB1, and CEI. The negative effect of ANP was completely reversed by KT5823 (a specific inhibitor of cGMP-dependent protein kinase), while C-ANP-(4-23) (an analogue of ANP and specific binder for natriuretic peptide receptors-C) was ineffective in oocyte maturation. Neither ANP nor C-ANP-(4-23) had an effect on spontaneous porcine oocyte maturation and cumulus expansion. These results suggested that ANP negatively regulates FSH-activated porcine oocyte meiotic resumption, meiotic maturation and cumulus expansion. The function of ANP on porcine oocyte maturation is via the cGMP dependent protein kinase (PKG) pathway.

Nitric oxide (NO) is animportant biological messenger in the regultlation of tissue homneostasis. It exhibits a wide range of effects during physiolgical and pathophy siological prucesses Typical beneficial ploperties of N0 include the regulation of vascular tone, the protection of cells against apoptosis, the modulation of immune responses, and the killing of microbial patteogens. On the other hand, NO may causese severe vasodilation and myocardial depression during bacterial sepsis or act as a cytotoxic and tissue-damaging molecule in autoimmune diseases Mitogen-activated protein kinase (MAPK) is afamily of serine/threonine protein kinases that in mammalian widely distributed cells. MAPK cascade play pivotal rolesin gene expression, cell prdiferation, differentiation, neuronal survival and pro-grammned cell death under a variety of experimental conditions. MAPKs transduce the signal for the cellular respons to are extracelluar stress-es or stimuli. The rdation between them. however, has never been reviewed Based on Our-researches and 0ther reports in the field, we review their reciprocal regulatury functions.

调节原始卵泡形成、起始生长的信号目前仍知之甚少。一个重要的原因就是缺乏一个良好的研究模型。我们以妊娠13天昆明白小鼠胚胎卵巢为研究材料，经过5天的贴壁培养后，分别用牛血清（FBS）、无血清（ITS）和含有人卵泡刺激素（FSH-ITS）培养液继续体外培养到第19天，发现ITS组培养的胚胎卵巢卵泡发育要显著优于FBS组（P<0.01），如：培养至第7天时（P1，相当于出生日），卵泡数分别为295±18和206±17；培养至第13天时（P7），卵泡数分别为594±31和262±28；培养至第19天时（P13），卵泡数分别为371±25和50±11（Fig.1，2&4）。ITS处理组的绝大部分卵巢在培养早期（如：P5前）都形成了皮质-髓质样的卵泡生长模式，而FBS处理组超过半数卵巢不能形成皮质-髓质样的卵泡生长模式，FSH-ITS处理组和ITS处理组的胚胎卵巢卵泡发育并无显著差异（Fig.2，4&5）。

Nitric oxide (NO) plays an important role in many pathological processes. The present investigation was undertaken to ascertain the effects of NO at high level on porcine oocyte meiotic development. Cumulusenclosed oocytes (CEOs) and denuded oocytes (DOs) were obtained from the follicles (2-6mm diameter). Exogenous NO was provided with sodium nitroprusside (SNP). Control (0mm), 0.1mm, 1mm or 10mm SNP was introduced to the medium. Meanwhile, 0.1mm, 1mm and 10mm preincubated SNP was also added to the medium respectively. The cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation and degeneration were determined 44 h after incubation. SNP inhibited cumulus expansion and cumulus cells DNA fragmentation in a dose-dependent manner. Significantly fewer CEOs treated by any level of SNP resumed meiosis than control (p<0.05) and more CEOs exposed to 0.1mm or 1mm SNP were arrested at GV stage. Moreover, SNP increased the percentage of CEOs at metaphase I (MI) stage but decreased the percentage of CEOs at metaphase Ⅱ(MⅡ) stage. 0.1mm or 1mm SNP showed no influence on the proportion of germinal vesicle breakdown of DOs and only 10mm SNP reduced the proportion. SNP accelerated degeneration of both CEOs and DOs, especially at higher concentrations. DOs were more sensitive to SNP toxicity. Preincubated SNP showed no effect on cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation or degeneration at lower concentration, but higher concentrations had effect, especially on the viability of porcine oocytes. Taken together, high level of NO inhibits porcine oocyte meiotic maturation, especially the transition from MI to MII, by mediating the functions of cumulus cells.

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24h in a medium containing 4mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), Nomega-nitro-L-arginine methyl ester (L-NAME) or Nw-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and Larginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10-3M L-NAME (PB/ 0.01) or 10-3M L-NNA (PB/0.01) and the inhibition could be reversed by the addition of SNP (10-5M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10-7, 10-6, 10-5M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1/4mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1mM) could significantly delay GVBD during the first 5h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4mM L-arg and 1mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.