雌激素受体α和β亚型在文昌鱼神经系统、哈氏窝和性腺中的定位

用兔抗人ER-α和ER-β多克隆抗体对文昌鱼神经系统、轮器哈氏窝和性腺进行免疫细胞化学的定位研究。结果揭示幼年和成年两性不同发育时期文昌鱼在这些部位分布ER-α和ER-β蛋白。ER-α定位在端脑、中脑、后脑和神经管中大多数神经细胞核,少数在胞质及其突起和神经纤维,ER-β则定位在细胞质或细胞膜上,少数在核内。ER—α免疫阳性物质主要分布在哈氏窝下层的上皮细胞核,少数在上层细胞质,β受体则在上层细胞核。在性腺,ER-α分布在卵巢中卵原细胞和小生长期卵母细胞胞质与核仁,生发泡(核)显免疫阴性,在大生长期卵母细胞核膜和核仁的免疫阳性显著增强,成熟期则在卵细胞生发泡表达,ER-β免疫阳性物质分布在卵原细胞和早期卵母细胞质以及成熟卵细胞的卵被膜检测到,生发泡显免疫阴性。在精巢,这两种ER亚型均定位在精原细胞、初级与次级精母细胞和足细胞质,精子细胞在胞核,精子显免疫阴性。另外,双染结果还揭示ER-α和ER-β在上述部位多数共存于同一细胞,少数在不同细胞表达,且在细胞定位有不同。首次发现这两种雌激素受体亚型在文昌鱼有广泛分布,它们介导雌激素对文昌鱼神经内分泌组织的调节作用。α和β受体在靶细胞定位的不同,提示两者在介导雌激素信号路线和基因转录机制可能有不同生理作用。     

雌、雄激素受体在文昌鱼神经系统和哈氏窝的免疫识别

用雌、雄激素受体的多克隆抗体对不同发育时期的文昌鱼神经系统和哈氏窝进行免疫识别。结果显示,雌、雄激素受体免疫阳性识别发生在文昌鱼端脑和中脑的中部与后部,以及神经管的背面和中央,后脑则为免疫阴性反应。两种激素受体免疫阳性物多数分布在神经细胞核内及其神经纤维,少数在胞质、雌、雄激素受体免疫阳性识别还出现在文昌鱼哈氏窝基部的上皮细胞核内或胞质中。雌、雄激素受体在文昌鱼神经系统和哈氏窝的免疫识别部位与脊椎运行相类似。研究表明,性类固醇激素像在脊椎动物中那样,可对文昌鱼脑和哈氏窝进行反馈调节。     

芳香化酶在文昌鱼神经系统、哈氏窝和性腺特异性定位:原位杂交和免疫细胞化学研究

芳香化酶活性发现在脊椎动物脑、脑垂体和性腺中,但在文昌鱼脑和哈氏窝的组织特异性定位尚无可利用资料。本文用免疫细胞化学和原位杂交技术,首次发现芳香化酶活性组织特异性定位在幼年和性腺发育不同时期雌、雄文昌鱼神经系统(脑和脊髓)、轮器、哈氏窝和性腺中。芳香化酶蛋白和转录物在前脑、中脑、脊髓、轮器和哈氏窝十分丰富,而后脑、早期卵巢和精巢不够丰富;没有芳香化酶表达的部位是哈氏窝另两种细胞(不规则形细胞和带纤毛粘液细胞)以及成熟卵巢和精巢;芳香化酶免疫活性物质分布在胞质,核为阴性。芳香化酶在文昌鱼神经系统、哈氏窝和性腺的分布模式与低等脊椎动物中的分布模式极为类似,尤其是芳香化酶在脑内调节哈氏窝分泌活动的神经内分泌中枢表达,并形成类似脊椎动物的文昌鱼原始的脑-芳香化酶调节系统。这些结果有力地证明,文昌鱼脑和哈氏窝高水平的芳香化酶活性像在其它脊椎动物中一样,对局部介导睾酮芳香化起着关键作用,同时还可能影响脑-芳香化酶系统参与调节哈氏窝的分泌活动[动物学报49(6)：800～806,2003]。     

雌激素受体α和β亚型在文昌鱼神经系统、哈氏窝和性腺中的定位

用兔抗人ER-α和ER-β多克隆抗体对文昌鱼神经系统、轮器哈氏窝和性腺进行免疫细胞化学的定位研究。结果揭示幼年和成年两性不同发育时期文昌鱼在这些部位分布ER-α和ER-β蛋白。ER-α定位在端脑、中脑、后脑和神经管中大多数神经细胞核,少数在胞质及其突起和神经纤维,ER-β则定位在细胞质或细胞膜上,少数在核内。ER-α免疫阳性物质主要分布在哈氏窝下层的上皮细胞核,少数在上层细胞质,β受体则在上层细胞核。在性腺,ER-α分布在卵巢中卵原细胞和小生长期卵母细胞胞质与核仁,生发泡(核)显免疫阴性,在大生长期卵母细胞核膜和核仁的免疫阳性显著增强,成熟期则在卵细胞生发泡表达;ER-β免疫阳性物质分布在卵原细胞和早期卵母细胞质以及成熟卵细胞的卵被膜检测到,生发泡显免疫阴性。在精巢,这两种ER亚型均定位在精原细胞、初级与次级精母细胞和足细胞质,精子细胞在胞核,精子显免疫阴性。另外,双染结果还揭示ER-α和ER-β在上述部位多数共存于同一细胞,少数在不同细胞表达,且在细胞定位有不同。首次发现这两种雌激素受体亚型在文昌鱼有广泛分布,它们介导雌激素对文昌鱼神经内分泌组织的调节作用。α和β受体在靶细胞定位的不同,提示两者在介导雌激素信号路线和基因转录机制可能有不同生理作用。     

文昌鱼神经系统、哈氏窝和性腺GnRH受体mRNA原位杂交的研究

用地高辛标记的寡核苷酸探针对ＧｎＲＨ受体在文昌鱼（Ｂｒａｎｃｈｉｏｓｔｏｍａ ｂｅｌｃｈｅｒｉ）神经系统、哈氏窝和性腺中的定位和表达进行原位杂交研究。结果显示,神经系统中神经细胞、尤其是在中脑右侧延伸出与哈氏窝相接触的漏斗样结构中的神经细胞、哈氏窝上皮细胞以及雌雄性腺中生殖细胞都有ＧｎＲＨ受体ｍＲＮＡ杂交信号,信号物质分布于胞质,胞核为阴性。表明文昌鱼神经系统、哈氏窝和性腺都能合成ＧｎＲＨ受体,从     

GnRH在文昌鱼脑和哈氏窝的分布

用免疫细胞化学方法和抗哺乳动物ＧｎＲＨ抗体研究了ＧｎＲＨ免疫活性细胞在文昌鱼脑和哈氏窝的分布。ＡＰＡ免疫染色法证明了哺乳动物ＧｎＲＨ免疫活性结构分布在文昌鱼端脑前端和靠近中脑的背部,中脑中部,神经管和哈氏窝中。ＧｎＲＨ－ｉｒ胞体有多种类型,一种是大脑胞体,可见在端脑和中脑,另一种为中等和小型胞体,这些胞体聚集在中脑中部（相当于鱼类下丘脑）以及端脑。长的念珠状ＧｎＲＨ－ｉｒ神经纤维从端脑腹外侧面延伸     

神经肽Y和β-内啡肽样免疫阳性物质在文昌鱼神经系统和哈氏窝的分布

用免疫组织化学方法首次发现,神经肽Y(NPY)和β-内啡肽(β-Ep)样免疫阳性物质分布在文昌鱼神经系统和哈氏窝。NPY样免疫阳性神经元出现在端脑前部和中部、中脑前部和中部以及后脑,NPY样免疫阳性神经纤维在文昌鱼脑的各部分与神经元交错呈网状密集分布。神经管的背面与中部均可观察到NPY样免疫阳性神经元及其阳性纤维。β-Ep样免疫阳性神经元及其神经纤维定位在中脑前部和中部以及神经管,且分布范围明显小于NPY。文昌鱼哈氏窝也有NPY和β-Ep样免疫阳性物质分布。这些结果表明,NPY和β-Ep可能作为脑内的一种神经递质,像鱼类那样,参与调节文昌鱼哈氏窝促性腺激素分泌细胞的分泌活动,这为文昌鱼脑-哈氏窝复合体的密切关系提供新的形态学证据。     

神经肽Y和β—内啡肽样免疫阳性物质在文昌鱼神经系统和哈氏窝的分布

用免疫组织化学方法首次发现,神经肽Y（NPY）和β－内啡肽（β－Ep)样免疫阳性物质分布在文昌鱼神经系统和哈氏窝。NPY样免疫阳性神经元出现在端脑前部和中部、中脑前部和中部以及后脑,NPY样免疫阳性神经纤维在文昌鱼脑的各部分与神经元交错呈网状密集分布。神经管的背面与中部均可观察到NPY样免疫阳性神经元及其阳性性纤维。β－Ep样免疫阳性神经元及其神经纤维定位在中脑前部和中部以及神经管,且分布范围明显小于NPY。文昌鱼哈氏窝也有NPY和β－Ep样免疫阳性物质分布。这些结果表明,NPY和β－Ep可能作为脑内的一种神经递质,像鱼类那样,参与调节文昌鱼哈氏窝促性腺激素分泌细胞的分泌活动,这为文昌鱼脑－哈氏窝复合体的密切关系提供新的形态学证据。     

性类固醇激素及其受体在文昌鱼性腺和神经系统中的分布

首次用雌二醇,睾酮及孕酮受体的多克隆抗体在文昌鱼性腺,脑泡和神经管中进行免疫细胞化学定位研究,结果表明,不同发育时期的卵原细胞和卵母细胞中都存在雌激素,雄激素和孕激素受体,在小生长期,3种受体通常定位在卵原细胞和早期初级卵母细胞的胞质或核膜,在大生长期和成熟期,则定位在胞质和核质,在雄性,3种类固醇激素受体分布在不同发育时期的精原细胞,精母细胞和精子细胞（雌激素受体例外）中,受体阳性物定位在胞质和核,另外,用免疫细胞化学方法和性类固醇激素抗体对文昌鱼不同发育时期的性腺和神经系统进行研究,结果揭示,免疫阳性物存在于不同发育时期的卵巢和精巢中,在卵巢发育是上期,卵原细胞和卵母细胞的胸质和核仁膜对紫二醇,睾酮和孕酮抗体显示强或中等强度免疫阳性反应,在大生长期和成熟期,卵母细胞胞质和核仁膜对睾酮和孕酮抗体的免疫阳性反应明显减弱,而对雌二醇则显著增强,在雄性,随着精原细胞和精母细胞对孕酮抗体发生强的免疫阳性反应,在成熟期则对雌二醇抗体发生免疫阴性反应,精子始终为免疫阴性,本文研究首次发现,文昌鱼脑泡和神经管中存在哺乳动物神经旮体激素样免疫阳性神经细胞及其纤维,免疫阳性物分布在神经细胞胸质,核显阴性反应,这些结果为证明文昌鱼性类固醇激素参与调节性腺发育成熟和生殖内分泌调控轴的功能成熟提供重要的形态学新证据。     

促黄体素（LH）和人绒毛膜促性腺激素（hCG）在原索动物神经系统,哈 …

Immunocytochemical localization of the nervous system, Hatschek's pit and gonads in protochordata was carried out using two specific antibodies against human LH and hCG. The results indicate that there are two different kinds of LH- and hCG-like immunopositive cells in the brain vesicle and nerve tuber of amphioxus, and nerve ganglion in Styela plicata showed immuno-negative reaction to LH and hCG antibodies. At the same time, we found for the first time that LH- and hCG-like immuno-positive cells existed in the early gonads (ovaries and testis) in protochordata. Positive substance distributed in the cytoplasm, nucleoplasm and nucleolar membrane of oogonia as well as early spermatogenic cells in testis. Afterwards, this positive substance was still distributed in the cytoplasmic membrane, cytoplasm, nuclear membrane and nucleoplasm of oocyte during the development and maturation of ovary, and spermatid and spermatozoa showed immuno-negative reaction in testis. These results will provide a new mophological proof that LH and hCG may be directly involved in regulation of the gonadal development and maturation of protochordata.     

青岛文昌鱼精子运动特征及计算机辅助分析

青岛文昌鱼的精子在精浆中不运动,在与精浆等渗的生理溶液中也不运动,把精液稀释到过滤海水或人工海水后,精子运动被激活。文昌鱼新鲜精液中精子浓度为(9.4±1.6)×1010/ml;在人工海水中,运动精子占总精子数的85.8%±6.9%,精子运动持续的平均时间是22.7±2.6min。文昌鱼精子经人工海水激活0.5min后,可观察到四种运动方式:(1)向前直线运动,其平均速度93.6±23.7μm/s,这类精子所占的百分比为27.8%±3.1%;(2)弧形曲线运动,平均速度55.6±18.9μm/s,所占的百分比为44.3%±2.5%;(3)左右摆动,平均前进速度27.4±13.4μm/s,所占百分比为14.7%±1.8%;(4)运动速度小于5μm/s的精子,视为不运动的精子,所占百分比为13.2%±1.8%。随着激活时间的延长,文昌鱼精子的运动状态发生改变,向前直线运动和弧形曲线运动的精子逐渐减少,而左右摆动和不运动的精子逐渐增多。     

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus,Branchiostoma belcheri

The immunohistochemical localization of nine different neuropeptides was studied in the central nervous system of the amphioxus, Branchiostoma belcheri. In the brain, perikarya immunoreactive for urotensin I and FMRFamide were localized in the vicinity of the central canal. One of the processes of each of these perikarya was found to cross the dorso ventral slit-like lumen of the central canal. Oxytocin-immunoreactive short fibers, but not perikarya, were detected in the ventral part of the brain. Perikarya immunoreactive for arginine vasopressin/vasotocin, oxytocin and FMRFamide were widely distributed in the spinal cord. Arginine vasopressin/vasotocin-immunoreactive fibers often made contacts with Rohde cell axons. Angiotensin II-immunoreactive perikarya were observed in the posterior half of the spinal cord, and urotensin I-immunoreactive perikarya were found in the caudal region of the spinal cord. Cholecystokinin/gastrin-immunoreactive fibers, but not perikarya, were detected in the spinal cord; some extended as far as the ependymal layer of the cerebral ventricle. No colocalization of the peptides examined was observed. No immunoreactivity for atrial and brain natriuretic peptides nor for urotensin II was detected. The present study indicates that there are at least six separate neuronal systems that contain different peptides, respectively, in the central nervous system of the amphioxus. Their functions remain to be determined.Part of this investigation has previously been presented in abstract form (Uemura et al. 1989)     

通过Cyt b基因同源序列比较评估厦门文昌鱼的分类学地位

白氏文昌鱼Branchiostomabelcheri (Gray)在我国和日本沿海均有分布 ,由于南、北方文昌鱼形态学上有一定差异 ,且二者间存在一些过渡类型 ,其分类地位问题仍有待进一步澄清。本文测定了厦门欧厝海域产的文昌鱼mtDNACytb基因序列 ,并与日本产的文昌鱼以及另外产于大西洋的两种文昌鱼Cytb基因序列比较。分子系统学分析结果表明 :厦门欧厝海域产的文昌鱼与日本产的文昌鱼平均遗传距离为 2 1 12 % ,达到了种间分化的水平 ;经过对已有文献和文昌鱼地理分布的综合分析 ,作者建议将原来的白氏文昌鱼青岛亚种B belcheritsingtauense提升为种 ,南、北方所产文昌鱼分别作为两个独立的种存在 ,即南方的B belcheri (Gray)和北方的B tsingtauenseTchangetKoo     

Temporal and spatial dynamics of amphioxus population (Branchiostoma belcheri tsingtaneuse) and its influential factors in Luan River Estuary,China

Population distribution of amphioxus (Branchiostoma belcheri tsingtauense) was investigated in the Luan River Estuary from 1999 to 2011, to describe its trends and discover the factors that influence its decline. Amphioxus are distributed on the seabed at 5–10 m depth, between the Xinkai Estuary and the Dapu River Estuary, where the primary sand components of the sediment are medium and fine, with particle size ranging from 0.001 to 1 mm. In recent years, the population density and biomass of amphioxus have sharply decreased. Changes in sediment granularity composition may significantly influence amphioxus distribution. Our data showed that the highest density region of amphioxus occurred where >90% of the sediment particles were between 0.063 and 0.5 mm in size. A reduction in sediment discharge from Luan River and the expansion of raft‐breeding mariculture may be causing the decline in amphioxus through habitat destruction.