Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio)

Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise. (c) 2007 Published by Elsevier B.V.

色林错裸鲤性腺发育的组织学研究

：采用组织切片法，对西藏特有鱼类色林错裸鲤的性藤发育、产卵时间和产卵类型等进行了研究。其性豫 的发育可分为六个时期，卵母细赡的发育分为5个时相。在第2时相晚期到第3时相早期．卵母细胞中具有 卵黄棱和校仁物质外排现条，核仁排出物可能与棱周的颗粒卵黄形成有关。第2时相到第4时相早期．核膜 外侧具有一透明层。产卵后来排出的成熟卵粒通过颗粒细胞吸收和利用。已达性成熟年龄的个体并不是每 年都参与繁殖活动，具有繁殖间隔现象。产卵时简始于4月上旬，持续到8月上旬。色林错裸鲤属于分狄同 步产卵类型。

Identification and characterization of a novel splice variant of gonadotropin alpha subunit in the common carp Cyprinus carpio

In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.

Induction of triploidy in large yellow crocker Pseudosciaena crocea (Richardson, 1846): effects of pressure shocks and growth performance in the first rearing year

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow-cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 degrees C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P < 0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).

硬壳蛤性腺发育和卵母细胞成熟机理初步研究

采用体外药物诱导的方法，研究了5-羟色胺（5-hydroxytryptamine，5-HT）诱导的硬壳蛤卵母细胞成熟过程中cAMP信号通路的作用。结果表明，5-HT （0.01—100µM）均能够显著地诱导硬壳蛤卵母细胞的成熟。磷酸二酯酶抑制剂—咖啡因、茶碱和IBMX（3-异丁基-1-甲基黄嘌呤）可以单独抑制卵母细胞的自发成熟，但效果不显著。10mM的咖啡因和茶碱以及5mM的IBMX能够显著地抑制5-HT的诱导效果。dbcAMP（双丁酰基环腺苷一磷酸）不但能够抑制卵母细胞的自发成熟，而且还可以抑制5-HT诱导的成熟。因此，cAMP信号通路参与了5-HT诱导的硬壳蛤卵母细胞的成熟过程，并且该信号通路起着负调控的作用。 研究了PLC（磷脂酶C）和PKC（蛋白激酶C）的激活剂/抑制剂对5-羟色胺诱导的卵母细胞成熟的影响。高浓度的新霉素（PLC抑制剂）可以抑制5-HT诱导的卵母细胞的成熟，而DMBA（9,10-Dimethy-1,2-benzanthracene，9,10–二甲基胆蒽，PLC激活剂）则能够促进成熟。PMA（phorbol 12-myristate 13-acetate，佛波十四烷酸乙酸酯，PKC激活剂）能够抑制5-HT诱导的成熟，而Spingosine（PKC抑制剂）则可以促进卵母细胞的成熟。从而推测，5-HT诱导的卵母细胞成熟需要磷脂酰肌醇信号通路的激活。PLC浓度的降低能够抑制5-HT诱导的卵母细胞成熟；PKC浓度的降低则会促进卵母细胞的成熟。因此，在硬壳蛤卵母细胞的成熟过程中，PLC起促进的作用，DAG（二酰肌甘油）–PKC通路则起抑制的作用。 细胞外高浓度Ca2+能够促进硬壳蛤卵母细胞的成熟，Ca2+离子载体A23187也可以促进硬壳蛤卵母细胞的成熟。1-100µM异搏定（Verapamil，钙离子通道阻断剂）能够抑制卵母细胞的成熟，而100µM的Verapamil能够完全抑制其成熟。上述结果表明细胞外Ca2+对硬壳蛤卵母细胞的成熟是必需的，而且起到促进卵母细胞成熟的作用。三氟拉嗪（TFP，Ca2+与CaM结合的拮抗剂）能够抑制卵母细胞的成熟，高浓度的三氟拉嗪（1mM）能够完全抑制卵母细胞的成熟。说明CaM起到促进卵母细胞成熟的作用。可见，Ca2+通过与CaM的相互作用，共同起到促进硬壳蛤卵母细胞成熟的作用。 5-HT诱导成熟的卵母细胞可以完成受精过程，其受精过程以及幼虫发育情况与正常受精发育过程类似，没有显著差异。高浓度的新霉素可以抑制受精过程，而茶碱和咖啡因对受精没有影响。从而推测，磷脂酰肌醇信号通路参与了硬壳蛤卵母细胞的受精过程，而cAMP信号通路可能没有参与受精过程。 发现硬壳蛤的性腺发育与我国常见的双壳类如泥蚶相似。硬壳蛤卵母细胞中卵黄粒主要由线粒体、高尔基液泡、内质网和微吞饮泡形成。

牙鲆性腺分化发育及类固醇激素T和E2对性腺分化的影响

本文采用组织学手段研究了牙鲆性腺在分化、发育和成熟过程中的变化。然后，通过放射性免疫方法（RIA）测定了牙鲆仔稚幼鱼全组织匀浆液中的性类固醇激素—睾酮（T）和雌二醇（E2）的含量，并结合牙鲆血清中T和E2含量的年周期测定，从内分泌学水平探讨了T和E2在其性腺分化、发育和成熟过程中水平的变化规律。同时，采用高温和雌性激素对性腺未分化的普通和雌核发育牙鲆仔稚鱼进行诱导处理，获得了较高比例的雄性鱼/假雄鱼或100%雌性鱼；并研究了这些外界环境因子对牙鲆性腺分化、性别比率及体内T和E2水平的影响，藉此探讨了牙鲆性别决定与性腺分化的细胞学和内分泌学机制。 对牙鲆仔稚幼鱼性腺的组织切片观察发现，培育水温18~20℃下，孵化后第45天、平均全长<22.0±2.8 mm的牙鲆，其性腺分化尚未开始，属于原始性腺；在孵化后70日龄、平均全长为38.0±1.7 mm左右，部分个体中观察到卵巢的雏形，其余个体的性腺在此阶段以及之后的一段时间内变化并不明显；到了第110天、平均全长达到86.5±5.9 mm时，雌性个体卵巢出现了卵原细胞向卵母细胞的转变，标志着卵巢分化的结束。在90日龄、平均全长为63.5±3.4 mm的雄性牙鲆中，精原细胞快速增殖，并观察到了输精管结构；进一步的细胞学分化则出现在100日龄、平均全长为76.0±8.6 mm的个体中，此时可以看到精小叶的形成；在平均全长为140.0±15.2 mm时，精巢中出现初级精母细胞，标志着性腺分化的基本完成。 对牙鲆仔稚幼鱼全组织匀浆和成鱼血清中的T和E2水平的比较发现，在全长为6 mm左右的仔鱼中T和E2含量均较高。随后，在性腺分化过程中T含量大大降低，E2的含量急剧增高，而性腺分化后期E2含量又降到较低的水平。在雄性牙鲆成鱼中， 从精巢第Ⅲ期开始，T含量随着精巢的发育而增加，到了精巢第Ⅴ期性腺发育成熟并排精后，又降低到较低的水平；E2含量在从精巢第Ⅲ期发育至精巢第Ⅴ期过程中略呈降低的趋势，但是总体上来说没有明显的差异。在雌性牙鲆成鱼中，卵巢从第Ⅱ期到第Ⅳ期的过程中，T水平逐渐升高，在第Ⅴ期时则明显降低；而E2含量在卵巢第Ⅱ期时保持较低的水平，随着卵巢的发育，E2含量逐渐增高，在卵巢第Ⅳ期时达到最高水平，在第Ⅴ期产卵后又有所降低。在雌雄个体中T和E2均呈现周期性的变化。5月份随着水温的升高，雄性个体T和E2含量显著上升；到了9月份又逐渐下降至最低值。雌性个体E2含量自3月份开始增高，在5月份急剧升高，并在6月份达到最高值；在7月份的时候，E2突然降低，而到了8月份又有所回升；9月份之后E2逐渐降低并在1月份左右降到最低；而T的含量分别在2月份和6月份出现两次高峰。 温度诱导牙鲆幼鱼性腺分化的结果表明，牙鲆中存在明显的TSD机制，即其性腺分化因饲育水温的不同而变化：在一定温度范围内，随着饲育温度的增加，牙鲆的雄性比例逐渐增高，常温对照组和21℃组中的雄性比例分别为51.62%、60.00%，而在24℃和28℃高温组中，雄性比例显著高于对照组，分别达到73.33%和87.27%。T和E2含量测定显示，在性腺分化时期，高温和对照组中T含量没有明显的变化，而温度处理组中的E2水平则低于对照组，特别是在28℃高温组，其E2水平显著低于对照组（P<0.05）。外源E2处理性腺未分化的牙鲆幼鱼的结果也表明，牙鲆的死亡率与雌性化比率均为雌性激素剂量依赖型的。随着外源E2剂量的增加，雌性比率增加，但同时死亡率也增高。此期间T和E2水平比较发现，在性腺分化时期，对照组中的T含量稍高于雌激素处理组；而对照组中的E2含量高于0.2 ppm和2 ppm两个低剂量组，却低于20 ppm和100 ppm两个高剂量组。 同时，还对人工诱导培育的雌核发育牙鲆和性反转牙鲆进行了性腺发育观察，在所观察的雌核发育牙鲆个体中，其雌性比例为83.33%，而高温28℃饲育群体中的雄性比例（即假雄鱼比例）为91.67%；在雌性个体中，也有一定比例的个体性腺发育不正常，有的性腺发育较小，有的则缺少部分性腺。进一步对雌核发育成体的血清中T和E2含量进行测量，发现在普通牙鲆个体中T含量显著低于雌核发育个体，而E2含量则高于雌核发育牙鲆；在雌核发育牙鲆中，性腺发育不正常的个体比性腺发育正常的个体中的T含量稍高，而E2含量则显著低于正常雌核发育牙鲆个体和普通牙鲆个体（P< 0.05）。

高原鼠兔的季节性繁殖

A nnual changes of the rep roduct ive act ivity in adult male p lateau p ika (Ochotona curzoniae) , a small endemic mammal in Q inghai2T ibet P lateau, w ere invest igated from J anuary to December, 1991. A ll of the animals w ere k illed and decap itated during the nigh t (23:00～ 24:00) and the p lasma, p ineal glands, testes ep ididym is, sem inal vesicles, deferent ducts were co llected and used for biochemical, and histo logical studies. Significant changes associated with seasonal cycles were found. (1) In February～ early April, the restoration phase, the weights of testes, epididym ides and deferent ducts were increased; the process of sperm atogenesis was strengthened and testo sterone level in plasma was increased, but the pineal weight and its melatonin content were decreased. (2) During the middle of April～ late May, the sexually active phase, a significant elevation of gonadal activity was observed. In this period, gonadalw eights were increased, spermatogenesis was completed, pineal weights were decreased and melatonin contents were fluctuated at alow level. These results suggested the increasing in sexual activity as well as in the ability of testo sterone secretion. (3) A striking reduction of test icular activity appears in June～A ugust. In this inhibition phase, gonada lweight, process of sperm atogenesis, plasma testo sterone level were decreased while the pineal weight and pineal melatonin content were increased. (4) During Sep tember～ J anuary, the sexually quiescent phase, declining in weights of testes and epididymides, arrest of spermatogenesis, decreasing of plasma testo sterone concent ration, fluctuating in pineal weights and increasing in pinealmelatonin level were observed. Our findings indicated that the male pikas under natural conditions exhibited an annual rep roductive cycle. A possible relationship between pineal activity and reproductive function was also suggested.