Rspo1在雌性脊椎动物性别分化中的功能

长期以来雌性脊椎动物的性别分化被认为是一个"默认"的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

小鼠性别决定过程中性腺体细胞分化的调控

哺乳动物的性腺由生殖细胞和体细胞共同形成,性别决定前的性腺具有双向分化的潜能,性腺中体细胞的分化决定其发育为睾丸或卵巢。这一分化过程受到多种因子的精细调控。其中SRY、SOX9、SOX3、SOX8、SOX10、FGF9/FGFR2、PGD2、AMH和DMRT1等参与睾丸的发育和分化,而FOXL2、CTNNB1、RSPO1、WNT4、Follistatin、ERα/β和BMP2则在卵巢发育过程中发挥关键作用。如果这些分子调控网络受到内源性或外源性因子的破坏,则会引起两性发育紊乱,甚至导致雄性向雌性或雌性向雄性的性别逆转。本文以小鼠模型为例,阐述了在性别决定过程中体细胞命运决定以及谱系分化的分子调控网络。     

脊椎动物性别决定和分化的分子机制研究进展

哺乳类性别决定是多种转录因子和生长因子相继表达和相互调控的结果。SRY的表达启动雄性通路并诱导下游雄性特异基因SOX9、AMH等的表达。FOXL2在雌性未分化性腺表达,WNT-4和DAX1也在雌性性别决定或分化时期表达,表明雌性通路也是受特定基因调控的,而并非“默认通路”。鸟类的性别也是由遗传基因决定的,EFT1(雌性)和DMRT1(雄性)可能是性别决定候选基因。爬行类为温度性别决定的典型,温度可能通过调节雌激素水平和控制性别特异遗传基因表达决定性别。大部分两栖类性别受环境因素影响,但发现DMRT1和DAX1可能与其精巢发育有关。鱼类性别决定和分化方式差异很大,多种因素(遗传基因、环境因素、类固醇激素等)参与了这一过程。从青Q鳉Y染色体定位克隆的DMY,被认为是第一个非哺乳类脊椎动物雄性性别决定基因。所有这些表明脊椎动物性别决定和分化机制是多样化的。     

高糖通过下调FoxO1磷酸化诱导β细胞去分化

胰岛β细胞发生去分化现象是导致其功能减退的机制之一。已有研究证明,FoxO1与β细胞去分化密切相关。然而,高糖是否可通过FoxO1诱导β细胞发生去分化目前尚未见报告。本研究通过不同浓度高糖干预MIN6细胞,采用葡萄糖刺激胰岛素分泌试验（GSIS）检测β细胞功能|实时荧光定量PCR及蛋白免疫印迹、免疫荧光方法检测高糖干预后β细胞内祖细胞标志基因、β细胞标志基因及FoxO1的表达变化。结果显示,不同浓度高糖干预β细胞后,当浓度达到35 mmol/L时,β细胞祖细胞标志基因表达明显增加。且在该浓度时,检测到β细胞标志基因表达明显降低,MIN6细胞葡萄糖刺激胰岛素分泌功能减退,磷酸化FoxO1表达减少。上述结果提示,高糖可诱导胰岛β细胞去分化的发生,其机制可能是通过FoxO1介导。     

Rspo1调控斑马鱼胚胎汇聚延伸运动的研究

Rspo1 (R-spondin 1)是分泌型Rspos (R-spondins)蛋白家族的成员,在雌性发育、血管生成和癌症等多个方面具有调控作用。为了研究Rspo1在早期胚胎发育中的功能,以斑马鱼(Danio rerio)作为模式生物,利用反转录PCR及原位杂交技术检测rspo1基因的时空表达模式;通过显微注射rspo1 mRNA或rspo1反义寡核苷酸(Morpholino, MO)对rspo1进行过表达或敲降;通过形态观察及原位杂交技术检测胚胎汇聚延伸(Convergence and extension, CE)运动是否正常;利用荧光素酶活性检测实验测定Wnt/PCP信号通路活性水平;通过蛋白印迹法检测表征Wnt/PCP信号通路活性的磷酸化JNK (Jun N-terminal kinase)蛋白的水平。结果显示:rspo1为母源基因,在12hpf前胚胎中呈全身性表达, rspo1的过表达或敲降均影响胚胎的CE运动;过表达rspo1降低Wnt/PCP信号通路报告质粒的活性,而敲降rspo1则增加其活性,与之相一致, rspo1敲降的胚胎中磷酸化JNK的水平显著升高;此外, rsp...     

罗非鱼性别决定及分化的研究进展

对罗非鱼的性别决定和分化的研究现状和研究成果进行综述,并探讨了在罗非鱼性别决定研究上的研究趋势。     

高等植物性别分化研究的某些进展

高等植物性别分化研究的某些进展邵宏波（四平师范学院生物工程研究室吉林四平１３６０００）关键词高等植物,性别分化,基因表达ＳＯＭＥＡＤＶＡＮＣＥＳＩＮＴＨＥＳＥＸＵＡＬＤＩＦＦＥＲＥＮＴＩＡＴＩＯＮＲＥＳＥＡＲＣＨＯＦＨＩＧＨＥＲＰＬＡＮＴＳ￥Ｓｈａｏ...     

苦瓜性别分化的特异蛋白质研究

对苦瓜（ＭｏｍｏｒｄｉｃａｃｈａｒａｎｔｉａＬ．）的两性期和雌、雄花早期发育过程中的３个时期花蕾,用毛细管电泳法进行可溶性蛋白质分析,发现了一些与性别分化有关的特异蛋白质。其中１１ｋＤ的蛋白质从幼雌花时期开始出现后,在雌花发育的３个典型时期都存在,很可能是雌花分化程序表达中的一种“关键蛋白”。与之类似,３０ｋＤ的蛋白质很可能是雄花程序表达中的一种“关键蛋白”     

南蛇藤雌性性别相关的RAPD和SCAR标记

南蛇藤(Celastrus orbiculatus Thunb)属卫矛科南蛇藤属落叶藤本植物,是中国传统中药材,雌雄异株,少量雄全同株。由于目前在分子水平上对南蛇藤性别差异的研究较少,极大地限制了对其的开发利用。本研究利用RAPD分子标记对南蛇藤雌株、雄株和雄全同株进行了差异比较。100个引物中有5个引物(S127、S140、S148、S174及S111)在不同性别南蛇藤的基因组DNA中扩增到存在明显差异的条带。根据序列分析的结果将RAPD引物转化成特异性较强的SCAR引物后,仅引物S111扩增出一条雌性特异性条带。序列分析发现,该片段包含两个超过100个氨基酸的开放阅读框,其功能有待进一步的研究。     

β-联蛋白促进大鼠外周血间充质干细胞存活及扩增

本研究旨在探讨β-联蛋白在促进外周血间充质干细胞（PBMSCs）的存活和扩增中的作用。采取SD大鼠外周血,分离培养得到PBMSCs,随机将细胞分3组：未处理组（CON）、β-联蛋白激动剂组（WAY-262611,WAY)和β-联蛋白抑制剂组（XAV-939,XAV）。体外培养90 d,观察各组细胞增生和凋亡情况。应用Western 印迹和ELISA方法观察细胞衰老标记性蛋白质p16和p21表达情况,以及β-联蛋白信号通路分子表达情况。外周血经过3代培养,可获得纯度较高的PBMSCs,体外培养40 d即发生衰老和凋亡,但WAY-262611可延长细胞存活时间至90 d以上,增加其体外增殖率1.08倍（P<0.05）,明显提高细胞增殖能力（P<0.05）;使WAY组p21和p16蛋白含量较CON组减少72.1%和68.4%（两者均P<0.05）,较XAV组减低92.9%和93.3%（两者均P<0.05）。与CON组比较,WAY组衰老细胞数减少61.4%（P<0.05）,而XAV组则增加53.3%（P<0.05）;WAY组细胞凋亡率减少50.2%（P<0.05）,而XAV组细胞凋亡率增加35.7%（P<0.05）;WAY组β-联蛋白表达水平增加2.2倍（P<0.05）,而XAV组则降低77.7%（P<0.05）。WAY-262611可升高WAY组β-联蛋白磷酸化水平（包括p-Ser552 和p-Ser675）97%和79%（两者均P<0.05）,升高抗凋亡蛋白质Bcl2表达水平1.39倍（P<0.05）,降低凋亡蛋白Bax和胱天蛋白酶3表达67.6%和83.3%（两者均P<0.05）;而XAV-939则降低Bcl2蛋白表达水平69.1%（P<0.05）,升高Bax和胱天蛋白酶3表达水平1.27倍和1.02倍（两者均P<0.05）。β-联蛋白可能是影响MSCs存活和增殖的重要信号途径。这些发现可能有助于提高用于组织工程的PB-MSCs的体外生产。     

PGC-1β和SREBP-1c在猪脂肪细胞分化过程中的相互作用

为研究过氧化物酶体增殖物激活受体γ辅激活因子1β(PGC-1β)与SREBP-1c在猪前体脂肪细胞分化过程中的表达规律及其相互作用,分析二者功能上的联系,采用Western 印迹及细胞免疫荧光技术检测PGC-1β与SREBP-1c在猪脂肪细胞分化过程中的表达,shRNA干扰和免疫共沉淀技术分别探讨了PGC-1β对SREBP-1c的调节作用及2种蛋白质在体内的结合活性.结果显示,PGC-1β与SREBP-1c 蛋白的表达均随猪脂肪细胞分化逐渐增加,且在分化细胞的核和胞浆中均有分布. 干扰PGC-1β显著下调了SREBP-1c和脂肪细胞分化标记基因C/EBPα的表达(P<0.05),同时降低了细胞内甘油三酯的积累.免疫共沉淀证明,PGC-1β与SREBP-1c蛋白在猪脂肪细胞分化过程中存在结合作用. 以上结果表明,PGC-1β能够促进猪脂肪细胞分化并对SREBP-1c有调节和结合作用,推测二者的结合可能与其对脂肪细胞的分化调节机制相关,将对PGC-1β调控脂肪细胞分化的功能和机理研究提供新途径.     

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus)

In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.     

Germ cell nuclear antigen (GCNA1) expression does not require a gonadal environment or steroidogenic factor 1: Examination of GCNA1 in ectopic germ cells and in Ftz-f1 null mice

The germ cell lineage is first recognized as a population of mitotically proliferating primordial germ cells that migrate toward the gonadal ridge. Shortly after arriving at the gonadal ridge, the germ cells begin to initiate a commitment to gamete production in the developing gonad. The mechanisms controlling this transition are poorly understood. We recently reported that a mouse germ cell nuclear antigen 1 (GCNA1) is initially detected in both male and female germ cells as they reach the gonad at 11.5 days postcoitum (dpc). GCNA1 is continually expressed in germ cells through all stages of gametogenesis until the diplotene/dictyate stage of meiosis I. Since GCNA1 expression commences soon after primordial germ cells arrive at the gonadal ridge, we wanted to determine whether the gonadal environment was essential for induction of GCNA1 expression. By examining GCNA1 expression in germ cells that migrate ectopically into the adrenal gland, we determined that both the gonadal and adrenal gland environments allow GCNA1 expression. We also examined GCNA1 expression in Ftz-F1 null mice, which are born lacking gonads and adrenal glands. During embryonic development in the Ftz-F1 null mice, the gonad and most germ cells undergo apoptotic degeneration at about 12.5 dpc. While most of the germ cells undergo apoptosis without expressing GCNA1, a few surviving germs cells, especially outside the involuting gonad clearly express GCNA1. Thus, although the Ftz-F1 gene is essential for gonadal and adrenal development, induction of GCNA1 expression in germ cells does not require Ftz-F1 gene products. The finding that germ cell GCNA1 expression is not restricted to the gonadal environment and is not dependent on the Ftz-F1 gene products suggests that GCNA1 expression may be initiated in the germ cell lineage by autonomous means. Mol. Reprod. Dev. 48:154–158, 1997. © 1997 Wiley-Liss, Inc.     

Cells on the move: Modulation of guidance cues during germ cell migration

In Drosophila melanogaster the progenitors of the germ-line stem cells, the primordial germ cells (PGCs) are formed on the outside surface of the early embryo, while the somatic gonadal precursor cells (SGPs) are specified during mid-embryogenesis. To form the primitive embryonic gonad, the PGCs travel from outside of the embryo, across the mid-gut and then migrate through the mesoderm to the SGPs. The migratory path of PGCs is dictated by a series of attractive and repulsive cues. Studies in our laboratory have shown that one of the key chemoattractants is the Hedgehog (Hh) ligand. Although, Hh is expressed in other cell types, the long-distance transmission of this ligand is specifically potentiated in the SGPs by the hmgcr isoprenoid biosynthetic pathway. The distant transmission of the Hh ligand is gated by restricting expression of hmgcr to the SGPs. This is particularly relevant in light of the recent findings that an ABC transporter, mdr49 also acts in a mesoderm specific manner to release the germ cell attractant. Our studies have demonstrated that mdr49 functions in hh signaling likely via its role in the transport of cholesterol. Given the importance of cholesterol in the processing and long distance transmission of the Hh ligand, this observation has opened up an exciting avenue concerning the possible role of components of the sterol transport machinery in PGC migration.     

胰高血糖素样多肽-1拮抗2型糖尿病胰岛细胞凋亡损伤

胰高血糖素样多肽-1（glucogen like peptide 1, GLP-1）在胰岛素分泌过程中扮演重要角色,并在改善β细胞功能方面有着令人瞩目的效应,但有关其作用机制尚需更深入研究。本研究探讨GLP-1对2型糖尿病（type 2 diabetes mellitus, T2DM）大鼠模型胰岛细胞损伤的影响,观察GLP-1在T2DM大鼠胰岛细胞凋亡损伤机制中所发挥的作用。HE染色结果发现,糖尿病大鼠胰岛损伤。ELISA结果表明,糖尿病患者和糖尿病大鼠血清中GLP-1表达水平上调。放射免疫结果表明,GLP-1和谷氧还蛋白1（Grx1）促进HIT-T 15细胞分泌胰岛素,Cd抑制胰岛素的分泌。免疫组化结果表明,糖尿病大鼠GLP-1加药处理后,各组与糖尿病组相比,药物提高了Grx1和胰岛素表达水平,降低了胰高血糖素表达水平,同时降低了活性胱天蛋白酶3（caspase-3）的表达。本研究结果提示,GLP-1在肥胖T2DM大鼠胰岛细胞凋亡中起保护作用,同时可调节胰岛素和胰高血糖素水平,其机制可能与Grx1相关     

稳定转染MiR-499对P19CL6细胞向心肌细胞分化的影响

小鼠miR-499基因包含在心肌重链肌球蛋白Myh7b基因的第19内含子中,并且在心肌细胞中特异表达,然而其在心肌细胞中表达的生物学功能和意义尚不清楚.利用可体外分化为心肌细胞的P19CL6细胞建立稳定表达miR-499的细胞株对研究miR-499的生物学功能具有重要意义.根据小鼠miR-499基因序列,设计PCR引物...     

Hormonal Control of Sex Differentiation of Potentially Female Floral Buds of Lagenaria siceraria var. hispida In Vitro

In the present study, several kinds of phytohormones were used for the control of sex differentiation of the potentially female floral buds of Lagenaria siceraria var. hispida in vitro. It was shown that both GA3 and STS (silver thiosulphate) could effectively change the direction of sex differentiation of the potentially female floral buds in vitro. In the MS medium, supplemented with IAA, BA and ACC(1-aminocyclopropane-l-carboxylic acid) at l nmol/L, male flowers would be induced from the potentially female floral buds by the addition of GA3 (1–500μmol/L). Herein the male flowers were induced more effectively by GA3 within 5–-20 μmol/L but it was not as effective as STS. In the MS medium supplemented with IAA, BA and ACC at 1 nmol/L and with GA3 at 20 nmol/L more male flowers were differentiated from the potentially female floral buds with the addition of STS within 100–500 μmol/L. On the contrary, when the MS medium were supplemented with IAA and ACC at 1 nmol/L and with BA increased to 100 nmol/L more female flowers were differentiated from the potentially female floral buds, even with addition of 10–50 nmol/L of GA3.     

RP4 通过miR-183-5p.1上调FOXO1抑制乳腺癌细胞增殖

近年来,越来越多的证据表明,长非编码RNAs在肿瘤发生发展中发挥重要作用。位于12号染色体的长非编码RNA RP4-816N1.7（简称RP4）在乳腺癌细胞中的作用未见报道。我们通过实时荧光定量PCR证实,RP4在乳腺癌细胞中的表达量普遍低于其在正常乳腺上皮细胞MCF-10A中的表达量。RP4在MCF-7和MDA-MB-231中表达量分别比其在MCF-10A中的表达量下调21.57%和91.33%。过表达RP4可明显抑制乳腺癌细胞增殖。敲低RP4可显著增加乳腺癌细胞的增殖能力。生物信息学预测,RP4可能与miR-183-5p.1结合,且叉头蛋白O1(FOXO1)可能是miR-183-5p.1的潜在靶标。实时荧光定量PCR结果提示,RP4可下调miR-183-5p.1,而miR-183-5p.1也可下调RP4和FOXO1的表达。双荧光素酶报告基因结果证实,miR-183-5p.1可与RP4结合,下调其表达,也能与FOXO1 3′UTR结合,抑制其mRNA和蛋白质水平的表达量。最后,本文通过BrdU实验证实,RP4通过FOXO1抑制乳腺癌细胞的增殖。总之,RP4通过内源性结合miR-183-5p.1,上调FOXO1表达,进而抑制乳腺癌细胞增殖。