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).

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.

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.

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）。

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.