夏国良
促性腺激素诱导卵母细胞体外成熟的作用机制
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- 姓名:夏国良
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博士生导师
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学科领域:
海洋化学
- 研究兴趣:促性腺激素诱导卵母细胞体外成熟的作用机制
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夏国良, M Zhang, Y Tao, B Zhou, H Xie, F Wang, L Lei, L Huo, Q Sun and G Xia
Journal of Molecular Endocrinology (2005) 34, 459-472,-0001,():
-1年11月30日
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).
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夏国良, Zhongxian Lu a, Guoliang Xia a, *, Anne Grete Byskov b, Claus Yding Andersen b
Molecular and Cellular Endocrinology 164 (2000) 191-196,-0001,():
-1年11月30日
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.
Amphotericin B, Ketoconazole, FSH, Meiosis-activating sterol (, MAS), , Mouse oocyte meiosis
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夏国良, Yong Tao a, b, Zhuo Fu a, Meijia Zhang a, Guoliang Xia a, *, Jie Yang a, Huirong Xie a
Y. Tao et al./Molecular and Cellular Endocrinology 222 (2004) 93-103,-0001,():
-1年11月30日
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.
Nitric oxide, Pig, Follicle, Ovary, Corpus luteum, Meiosis
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夏国良, 王海滨, 李美玲, 李莹辉
To whom correspondence should be addressed. VoL. 9 No.7 (2000),-0001,():
-1年11月30日
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)。
mouse, fetal ovary, culture system, follicular development, FSH
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夏国良, Meijia Zhang a, Haibo Tang b, Guanchen Shen b, Bo Zhou a, Zhenlong Wu a, Zhenxin Peng c, Jinguo Zhang c, Jun Yan a, Guoliang Xia a, *
M. Zhang et al./Theriogenology xxx (2005) 1-12,-0001,():
-1年11月30日
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.
Giant panda, Spermatozoa, Atrial natriuretic peptide (, ANP), , Acrosome reaction, Zona pellucida
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夏国良, 王海滨, 夏国良*, 王群, 李美玲, 吕忠显
科学通报,2001,46(1):54~56,-0001,():
-1年11月30日
胚胎期生殖细胞和体细胞相互作用形成卵泡是一个复杂的生理过程。生殖细胞定向分化成卵母细胞后,必须以卵泡的形式存在才有可能完成最终的生长发育。因此,研究生殖细胞和体细胞体外重组形成卵泡的可能性具有重要的理论和实践意义。利用微滴培养法将胶原酶消化分离的12~16d胎龄的小鼠卵巢细胞分别培养,发现不同胎龄的卵巢细胞均能相互黏连形成多细胞聚集体;12~13d胎龄小鼠卵巢细胞体外不能形成卵泡;14~15d胎龄小鼠卵巢细胞体外经过4d培养后形成少量卵泡样复合体;16d胎龄小鼠卵巢细胞培养2d后形成大量具有典型卵泡特征的小卵泡.实验结果直接证实一定胎龄的小鼠卵巢生殖细胞和体细胞能够在体外重新构建形成卵泡;胎鼠卵巢生殖细胞获得诱导体细胞分化形成卵泡的能力是一个渐进过程.该研究为卵泡体外重组并重新构建卵巢进行卵巢移植提供了直接的实验依据。
小鼠, 胚胎卵巢, 体外重建, 卵泡
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夏国良, Huirong Xie a, Guoliang Xia a, *, Anne Grete Byskov b, Claus Yding Andersen b, Shumin Boa, Yong Tao a
H. Xie et al./Molecular and Cellular Endocrinology 218 (2004) 155-163,-0001,():
-1年11月30日
This study describes a model for short-term culture of intact mouse follicles under serum-free conditions. Follicles were either obtained b from immature mice receiving no ovarian stimulation (i.e. no eCG-primed protocol, group Ⅰ) or from mice undergoing ovarian stimulation (i.e. eCG-primed protocol, group Ⅱ). Follicles were grouped according to size (100-170, 180-200, 210-250, 260-350 and 360-400m, respectively) and cultured for 24h (group I) or for only 6 h (group II). Induced meiotic resumption of follicle-enclosed oocytes were evaluated following stimulation with gonadotropins (i.e. FSH and hCG), AY9944-A-7, an inhibitor of 14-reductase, and RS-21745, an inhibitor of lanosterol 14 -demethylase; both enzymes affect synthesis of the meiosis activating sterols (MAS) that induce oocyte maturation. The frequency of oocyte degeneration was also recorded. In group I, FSH (10-200 IU l-1) and AY9944-A-7 (5, 25 and 50M) separately induced resumption of meiosis in oocytes derived from follicles with a diameter of 180-400m. hCG (1.0 and 10 IU ml-1) exhibited a similar but weaker effect on oocytes present in follicles with a diameter of 260-400μm. Irrespective of follicular diameter oocytes obtained from follicles in group II responded to hCG and FSH by resuming meiosis. FSH (50 IU l-1) alone or hCG (10 IU ml-1) alone both increased the GVBD percentage of oocytes enclosed in follicles with a diameter 260-400m, but the response to hCG was not significant compared to control. FSH (50 IU l-1) combination with hCG (10 IU ml-1) showed an additive effect raising the rate of GVBD after 6h culture. Addition of 50 or 100M RS-21745 was able to attenuate gonadotropins-induced resumption of meiosis to below background levels. In conclusion, the ability of FSH to induce meiotic resumption of follicle-enclosed mouse oocytes is correlated to follicle size, being most pronounced in larger follicles. hCG caused a similar but less pronounced effect. The ability of RS-21745 to inhibit and the ability of AY9944-A-7 to enhance oocyte maturation of follicle-enclosed oocytes support the concept of FSH employing MAS as a downstream signal transduction molecule for initiation of oocyte maturation in mice.
Follicle-stimulating hormone, Human chorionic gonadotropin, Meiosis, Follicle-enclosed oocyte
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夏国良, Meijia Zhang, Yong Tao, Guoliang Xia*, Huirong Xie, Haiyan Hong, Fengchao Wang, Lei Lei
M. Zhang et al./Theriogenology xxx (2005) 1-15,-0001,():
-1年11月30日
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.
Atrial natriuretic peptide, Follicle-stimulating hormone, Pig, Meiosis, Signal transduction
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【期刊论文】Regulation between nitric oxide and MAPK signal transduction in mammals*
夏国良, TAO Yong, , ZHANG Meijia, HONG Haiyan and XIA Guoliang**
Progress in Natural Science Vol. 15 No.1 2005,-0001,():
-1年11月30日
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.
MAPK, nitric oxide, nitric oxide synthase, signal tpallsduotion.,
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夏国良, Zhongxian Lu, Guoliang Xia*, Jianchao Zhang
Molecular and Cellular Endocrinology 182 (2001) 225-232,-0001,():
-1年11月30日
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.
Protein kinase C, Protein kinase A, Mouse, Follicle stimulating hormone, Oocyte maturation
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