不同倍性长牡蛎闭壳肌和精子的组织学和超微结构观察

以二倍体、三倍体、四倍体长牡蛎(Crassostrea gigas)闭壳肌和精子为研究对象, 采用石蜡组织切片、透射电镜和扫描电镜方法, 比较了 3 种倍性长牡蛎闭壳肌和精子的组织学特性及超微结构的差异。研究结果显示, 二、三、 四倍体闭壳肌组织的肌纤维都包含粗肌丝、细肌丝、肌膜、肌束膜、肌内膜、线粒体、囊泡、小管、细胞核、肌浆网等结构, 但三者的形态及超微结构不同, 其中三倍体闭壳肌线粒体直径、肌纤维直径、粗肌丝密度显著大于二倍体和四倍体, 肌纤维密度显著小于二倍体和四倍体。不同倍性精子的扫描电镜和透射电镜结果显示, 二、三、四倍体长牡蛎精子在组成结构上相似, 分别由头部、中段和尾部构成, 但不同倍性精子中各结构大小和线粒体数目不同。在长牡蛎精子头部长度、宽度, 顶体高度、宽度, 线粒体长度的测量分析中, 四倍体精子的头部、顶体以及线粒体显著大于二、三倍体。三倍体精子的头部和顶体显著大于二倍体, 但三倍体精子的线粒体大小与二倍体相比无显著差异。此外, 二、三倍体精子均含 4 个线粒体, 但在本研究中发现了二倍体精子含有 5 个(占比 1%)线粒体的特例。四倍体长牡蛎精子的线粒体数目不同, 含有 4 个(占比 46%)、5 个(占比 52%)、6 个(占比 2%)这 3 种情况。 该研究阐明了不同倍性长牡蛎闭壳肌及精子的组织学特性和超微结构, 为深入研究长牡蛎肌纤维生物学特性和精子繁育生物学提供了基础资料。     

星突江鲽精子的超微结构

应用扫描电镜和透射电镜观察了星突江鲽Platichthysstellatus精子的超微结构。星突江鲽的精子由头部、中段和尾部组成。头部呈球形，主要结构为细胞核。核前端无顶体，后端有植入窝，植入窝较浅，凹入深度约为细胞核直径的I／4。核中染色质致密，但存在染色质较浅的头部凹陷和核泡。中段包括中心粒复合体和袖套。近端中心粒的长轴与基体的长轴相互垂直，呈“T”字型排列，基体的长轴与精子的长轴平行。袖套很浅，其最深处只达到线粒体下端的1／3处。在袖套线粒体的外面，具有大量的椭圆形囊泡存在。尾部细长，主要由轴丝构成，为典型的“9＋2”微管结构。鞭毛较粗，长有两排侧鳍，侧鳍不对称。鞭毛细胞质中可见大小不一的椭圆形囊泡存在，囊泡的分布不均匀，一侧囊泡数量较多，另一侧偏少。鞭毛的这种结构与精子的运动相适应。     

Mucus piRNAs profiles of Vibrio harveyi-infected Cynoglossus semilaevis: A hint for fish disease monitoring

The half-smooth tongue sole, Cynoglossus semilaevis, is an important cultured flatfish species. Vibrio harveyi is a common pathogen to this fish, which may result in great economic loss to C. semilaevis culture industry. piRNAs, a non-coding RNAs with 26–32 nt, have been regarded as promising biomarkers for cancer diagnosis and fish diseases. Here, we extracted the RNA from mucus of C. semilaevis and constructed the differential expression profiles of piRNAs between the sick fish (MS) and healthy fish (MC). We identified 45,696 differentially expressed piRNAs including 22,735 up-regulated piRNAs and 22,961 down-regulated piRNAs in MS group compared with MC group. The GO enrichment and KEGG pathway enrichment analyses of the differential piRNAs were carried out. The result showed immunity-related target genes mainly involved in immune system process, response to stimulus, cell killing, immune system, infectious diseases and cell growth and death. The 10 most differentially expressed piRNAs were chosen to perform the qRT-PCR, while only seven piRNAs were consistent with the sequence result. Compared with MC group, the expression levels of piR-mmu-72173>piR-rno-62831>piR-xtr-704880, piR-dme-15546979, piR-mmu-49941660, piR-mmu-29283297 and piR-mmu-1758399 were significantly lower, and piR-gga-10574 and piR-gga-134812 were significantly higher in MS group. These piRNAs may be potential biomarkers during the V. harveyi infection of C. semilaevis. This study could provide a new method to identify the infection status of C. semilaevis and understand better about the innate and adaptive immune system in C. semilaevis during bacterial infection.     

嘉庚蛸精子发生的超微结构

应用透射电镜技术研究嘉庚蛸精子发生过程中各阶段细胞的形态、细胞器及细胞核的动态结构变化。精原细胞呈椭圆形,细胞质少、核大,有线粒体、高尔基体等细胞器;初级精母细胞呈圆形或卵圆形,线粒体多,索条状内质网发达,高尔基体常集中分布,中心粒形成;次级精母细胞呈圆形、椭圆形或不规则形,线粒体嵴发达、基质丰富,高尔基囊泡积累电子致密物,中心粒发出的“9＋2”轴丝结构形成。精细胞分化过程分为6个时期,主要特点为：①核内染色质凝集经颗粒化、纤维化及片层化,最后,形成高电子密度的均质结构;与此相应,核形态由椭圆形、变成长椭圆形、再变成细长柱状,核后端最后向前凹入形成核内沟;精子核的形态建成为染色质的凝集与核周微管加压协同作用的结果。②高尔基囊泡积累电子致密物、融合发育成顶体囊,逐渐延长演变成钻头形的顶体。③线粒体向核后端迁移,形成精子尾部的线粒体鞘。     

Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga,Brycon nattereri

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS^? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO₃ (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.     

云斑尖塘鳢精子的超微结构

采用扫描电镜和透射电镜观察了云斑尖塘鳢(Oxyeleotris marmoratus)的精子结构。结果显示:扫描电镜下云斑尖塘鳢精子由头部和尾部(鞭毛)组成。头部近似圆形,直径1.98～2.12μm,尾部鞭毛长23.22～32.92μm。透射电镜下云斑尖塘鳢精子由头部、中段和尾部三部分组成。头部细胞核呈扁卵圆形,前端不具顶体,染色质致密,细胞核内偶见小空隙;核膜与质膜间有间隙,其间分布有细胞质、囊泡、溶酶体,在细胞核后端,还分布有5～7个呈环形单层排列的线粒体;植入窝较浅,中段不明显,包括中心粒复合体和袖套;精子尾部的鞭毛细长,轴丝为典型的"9+2"型结构;轴丝外侧具有较发达的侧鳍。     

An experimental study on the compensatory growth of tongue sole,Cynoglossus semilaevis (Günther, 1873), following lower temperature manipulation

The compensatory growth, body composition and energy budget of juvenile tongue sole, Cynoglossus semilaevis (Günther, 1873), subjected to an unfavourable temperature were investigated during 56 days experiment. Fish were divided into four groups including three groups, which were reared at 16°C for 1, 2 and 3 weeks, respectively, then returned to 22°C (recorded as A1, A2 and A3) and a control group (continuously reared at 22°C, C). At the end of the experiment, the body mass was significantly higher in A1 than that in the control (P < 0.05), which indicated over compensatory growth occurred in A1 fish. No significant difference was found in body mass among A2, A3 and C fish (P > 0.05), which indicated complete compensatory growth occurred in A2 and A3 fish. The underlying mechanisms for compensatory growth could be attributed to an improved energetic efficiency resulting from reduced metabolic expenditure and higher feed efficiency during the period of recovery. Furthermore, the fish exposed to lower temperature for 3 weeks showed hyperphagia. The results suggested that the juvenile C. semilaevis exposed to lower temperature for a suitable period (1–3 weeks) used feed more efficiently. This approach may be a useful rearing strategy for indoor culture of C. semilaevis.     

虾夷扇贝精子形态结构和超低温冷冻损伤的电镜观察

应用透射电镜和扫描电镜技术,研究了超低温冷冻保存前后虾夷扇贝精子超微结构和形态的变化。结果显示,虾夷扇贝精子由头部、中段和尾部3部分组成,外被光滑质膜;顶体位于头部最前端,呈倒"v"形,细胞核近似圆柱状,电子密度较高;4个线粒体和两个相互垂直的中心粒构成了精子的中段;鞭毛细长,轴丝为典型的"9 2"结构。经超低温冷冻保存后,冷冻损伤的精子表现为:精子被膜肿胀、被膜与核膜分离、丢失;顶体破裂、内容物流出;线粒体解体、线粒体嵴变形;鞭毛被膜肿胀、部分精子鞭毛脱落。可以推测,超低温冷冻保存对精子膜、顶体、线粒体和鞭毛的损伤可导致冻精活力和受精能力的下降。     

半滑舌鳎CD40基因克隆及其免疫功能分析

本研究通过RACE技术在半滑舌鳎(Cynoglossus semilaevis)中克隆得到1个CD40同源基因。该基因c DNA全长为2098 bp,开放阅读框为1011 bp,可编码由336个氨基酸组成的蛋白质。推导得到的CD40氨基酸序列包含1个信号肽、1个跨膜结构区、2个N-糖基化位点以及含4个富含半胱氨酸的保守结构区。不同物种氨基酸序列同源分析结果显示,该基因与哺乳类、两栖类以及硬骨鱼类相似性为28%~47%,其中与硬骨鱼类相似性较高。系统进化分析结果显示,半滑舌鳎CD40与其他硬骨鱼类CD40聚类为一支,哺乳类与两栖类CD40聚类为另一支。荧光定量PCR结果显示,CD40在健康半滑舌鳎不同组织中均有表达,其中在鳃、肝和脾等组织中表达量较高,在肠、肌肉和皮肤中表达量较低,在脑和肾中微量表达。经哈维氏弧菌(Vibrio harveyi)感染刺激后,与PBS处理的对照组相比,该基因m RNA在半滑舌鳎3个主要免疫组织(肝、脾和肾)中表达量均呈显著性升高,在肝中12 h到达峰值,在脾和肾中6 h达到峰值。上述结果表明CD40基因参与了半滑舌鳎抵抗哈维氏弧菌的免疫应答反应。     

九孔鲍精子发生的超微结构

利用透射电镜观察九孔鲍（Haliotis diversicolor aquatilis）精子的发生过程。结果表明,九孔鲍精子的发生历经精原细胞、初级精母细胞、次级精母细胞、精细胞和成熟精子5个阶段。其中精细胞的分化可分为5期,主要特点为：Ⅰ期,一些高电子致密颗粒参与顶体的形成;Ⅱ期,核形态由椭圆形到近圆形,再逐渐拉长为长圆柱状;Ⅲ期,核染色质以颗粒状形式凝集,最后向高电子密度均质状态转变;IV期,线粒体逐步融合、体积增大和嵴发达;Ⅴ期,中心体移动及鞭毛形成,胞质逐渐减少。成熟精子为原生型,由头部、中段和尾部组成。通过观察精子发生过程中细胞形态结构的变化,分析了其演变规律,同时与软体动物的其他种类作了比较研究。     

Influence of larval co-feeding with live and inert diets on weaning the tongue sole Cynoglossus semilaevis

The tongue sole Cynoglossus semilaevis, an inshore fish in China, has showed great potential in aquaculture recently. However, poor survival was recorded during the period of weaning from live Artemia to artificial diets. In this paper, the influence of co‐feeding larvae with live and inert diet on weaning performance was described. The C. semilaevis larvae were reared at 21 ± 1 °C and fed four different feeding regimes from 6 days post‐hatching (dph): A, Artemia (10 individuals mL^?1); B, Artemia (5 individuals mL^?1); C, mixed diet (10 Artemia individuals mL^?1 and 12 mg L^?1 inert diet); and D, mixed diet (5 Artemia individuals mL^?1 and 12 mg L^?1 inert diet). Rotifers were also supplied in all cases during the first days of feeding. Mixed diets of commercial formulated feed and live prey (rotifers and Artemia) allowed larvae to complete metamorphosis, achieving similar specific growth rate (SGR) (18.5 ± 1.4% and 18.7 ± 1.6%) and survival (40 ± 7.6% and 48.5 ± 6.8%) compared with larvae fed on live feed alone (SGR of 18.3 ± 1.2%, 19.3 ± 1.9% and survival of 41.2 ± 11.3%, 38 ± 4.9%). However, in metamorphosed fish, when live feed was withdrawn on 31 dph, there was significant difference (P < 0.05) in survival and growth among treatments. Metamorphosed fish, previously fed mixture diets during larval stages, had similar survival (62.1 ± 7.6% and 62.8 ± 3.9% for regimes C and D, respectively) but higher than that obtained for fish that previously fed on live feed (49.3 ± 2% and 42.1 ± 3.9% for regimes A and B, respectively) after weaning (day 60). The SGR of weaned fish previously fed live feed was similar (3.1 ± 0.6% and 2.92 ± 0.6% for regimes A and B, respectively) but lower than that recorded for fish that was fed from day 6 to day 30 on the mixed diet (4.5 ± 1.1% and 4.9 ± 0.3% for regimes C and D, respectively). It is suggested that weaning of C. semilaevis from early development would appear to be feasible and larval co‐feeding improves growth and survival.     

Progestin is important for testicular development of male turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>) during the annual reproductive cycle through functionally distinct progestin receptors

In teleost, sex steroid hormones are critical for reproduction. Progestin is known to promote spermiation. To further understand the functions of progestin via its receptors during the annual reproductive cycle in male turbot (Scophthalmus maximus), we observed testicular development, quantified several sex steroid hormones, detected the expression of progestin receptors, and measured various sperm parameters. Results showed that the turbot testicular structure was of the lobular type. During breeding season, a number of spermatocytes (stage III) developed into spermatids (stage IV), then differentiated into sperm during spermiogenesis (stage V), and finally regressed to spermatocytes (stage VI). Concomitant with testicular development, serum progesterone (P4) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) exhibited higher levels from stage IV to V than other stages. Furthermore, males with higher motility sperm showed higher levels of P4 and DHP compared with fish with lower motility sperm. These results indicated that P4 and DHP might induce spermatogenesis due to seasonal changes. Concurrently, in testes, the nuclear progesterone receptor (pgr) was expressed throughout the reproductive cycle and its level peaked during spermiogenesis while expression of membrane progestin receptor alpha (mPRα) did not change significantly. However, in sperm, mPRα expression was higher than in testes and had a significant positive correlation with curvilinear velocities (VCL), sperm motility, and motility duration. In conclusion, progestin appears to exert a direct pgr-mediated effect on spermiogenesis and improve sperm motility characteristics depending on the abundance of mPRα protein in sperm during spermiation.     

Effects of dietary n‐3 to n‐6 fatty acid ratios on spawning performance and larval quality in tongue sole Cynoglossus semilaevis

This study investigated the effect of n‐3 to n‐6 fatty acid ratios in broodstock diets on reproduction performance, fatty acid composition of eggs and gonads of tongue sole Cynoglossus semilaevis. Broodstock were fed five isonitrogenous and isoenergetic diets for 60 days. The supplemented lipids were prepared by a combination of fish oil and soybean oil inclusion FO (fish oil); FSO1 (fish oil: soybean oil = 7:1); FSO2 (fish oil: soybean oil = 2.2:1); FSO3 (fish oil: soybean oil = 1:1); FSO4 (fish oil: soybean oil = 1:4.3) as lipid sources with different n‐3 to n‐6 fatty acid ratios 10.40, 5.21, 2.81, 1.71 and 0.87. Results showed that relative fecundity, fertilization rate and survival rate of larvae at 7 days posthatching were all higher in broodstock fed FSO1 and FSO2 diet and significantly (P < 0.05) decreased in groups fed FSO3 and FSO4 diets. The best result in starvation tolerance test was obtained in FSO2 diet. The present study suggests that n‐3 and n‐6 PUFA ratio in broodstock diet has a considerable effect on spawning performance, egg and larval quality for C. semilaevis.     

半滑舌鳎溃疡病病原菌的分离、鉴定及其致病性分析

采用2216E和TCBS培养基分别从患溃疡病半滑舌鳎的肠道和体表病灶分离获得菌株40株，经血平板检验分离菌株的溶血活性，体外筛选并鉴定出3株潜在致病性菌株，即哈维氏弧菌、溶藻弧菌以及副溶血性弧菌。通过将3株潜在致病菌与健康半滑舌鳎的肠道上皮细胞共培养，检测病原菌刺激后半滑舌鳎肠道上皮细胞相关免疫基因的相对表达量及共培养后肠道上皮细胞的凋亡率，对病原菌的致病性进行细胞水平的筛选。结果显示，经哈维氏弧菌刺激后肠道上皮细胞白介素10基因(IL-10)表达量极显著上调，说明哈维氏弧菌引起的共培养上皮细胞免疫反应最为剧烈，且哈维氏弧菌与肠道上皮细胞共培养后导致细胞的凋亡率高达48.3%，其次为溶藻弧菌(36%)和副溶血性弧菌(34.5%)，推测哈维氏弧菌为半滑舌鳎溃疡病高致病性弧菌。通过对半滑舌鳎进行浸浴回感实验，结果发现，哈维氏弧菌、溶藻弧菌和副溶血性弧菌人工回感后第6天半滑舌鳎的累计死亡率分别为100%、95%和75%，与细胞水平检测结果相吻合。实验表明，所筛选到的3株弧菌均具有较强的毒性和致病性，尤其以哈维氏弧菌致病性最强，并由此推测半滑舌鳎溃疡病可能由多种致病菌共同感染所致。     

刺参精子发生的超微结构研究

利用透射电镜观察刺参的精子发生过程,揭示其从精原细胞到精子的一系列超微结构变化。根据顶体发育的特点,将刺参精细胞发育分为精原细胞、精母细胞、精细胞等3个发育时期。精原细胞体积最大,有明显核仁,出现前顶体颗粒。初级精母细胞略小,染色质开始凝集,次级精母细胞开始出现尾部。精细胞时期顶体颗粒形成,线粒体明显的融合成一巨型线粒体。透射电镜和扫描电镜观察精子结构,成熟精子为原生型,由头部、中部、尾部组成。头部圆形,直径约3μm,围绕头的中部有一环形凹陷沟,尾部长约50~60μm。核染色质凝集成团块状,中部不发达,线粒体融合成1个环绕中部。尾部鞭毛横切面为简单的“9 2”型结构。     

褐牙鲆精子超微结构观察

利用扫描电镜和透射电镜对自然成熟的褐牙鲆精子超微结构进行了观察。褐牙鲆精子的头部近似圆形,直径1.49±0.17μm,尾部鞭毛较长,达40.17±0.65μm。褐牙鲆精子由头部、中段和尾部(鞭毛)构成。头部的顶端无顶体,细胞核为圆形,染色质致密。核膜分由内核膜和外核膜构成,其间有核周腔。核膜与质膜间有较大间隙,其间分布有许多细胞质、囊泡和溶酶体,在细胞核后端,核膜与质膜之间除细胞质外,还分布有5~6个呈环形单层排列的线粒体。植入窝位于细胞核后端的凹陷处,中心粒复合体位于植入窝内。基体的头端由电子致密物质构成,基体的末端与鞭毛起始端相连,鞭毛从袖套腔中伸出。鞭毛中心结构是轴丝,轴丝为"9+2"微管结构。轴丝外侧有侧鳍。     

Artificial gynogenesis in Cynoglossus semilaevis with homologous sperm and its verification using microsatellite markers

Effective methods for induction of gynogenetic diploids in Cynoglossus semilaevis are needed to initiate monosex culture. An effective protocol to induce half‐smooth tongue sole gynogenesis using homologous sperm was developed in this study. A UV dose of 50 mJ cm^?2 was found to be the most effective for genetic inactivation of tongue sole sperm. Treatment optima for cold shocks were 5 °C for 20–23 min at 5 min after fertilization and the hatching rate of gynogenetic diploids was 10.0%. Microsatellite analysis at locus Csou 6 revealed that there was no genetic contribution from the paternal genome in 24 progenies of a meiotic gynogenetic family. Polymerase chain reaction demonstrated that only four individuals of 24 meiotic gynogenetic diploids produced the female‐specific band of about 205 bp. The female/male ratio of gynogenetic diploids was significantly different from the theoretical ratio of 1:1. It is possible that there are some recessive lethal genes in W chromosome.     

A morphological classification proposal for curimba (Prochilodus lineatus) sperm damages after cryopreservation

The present study evaluated some cryoprotectants and concentrations for curimba (Prochilodus lineatus) semen preservation from a simple methodology of analysis of sperm morphology. Semen of nine males was diluted (1:4) into eight cryosolutions, all of them containing 5% of BTS^? Minitüb, still comprising methanol or dimethyl sulphoxide (DMSO) at four concentrations: 5%, 7.5%, 10% and 12.5%. Morphological sperm analysis was performed on an optical microscope at × 1000 magnification. Damages on the head, midpiece and tail rest (the main and terminal portions) were investigated and classified as primary or secondary, according to the assumed influence on fertilization. There was a linear reduction in the total damages with an increase in the cryoprotectant concentration. Samples with DMSO showed the lowest percentage of fractured tails and tail stumps. The cryopreservation protocol retained the fertilizing potential of the sperm after freezing and both methanol 5% and DMSO 7.5% conferred adequate protection to the curimba sperm cells. Sperm morphology, as pointed out here, must be incorporated into routine fish sperm (cryopreserved or not) analysis once sperm defects influence fertilization directly. To the best of our knowledge, this was the first report on curimba sperm damages after cryopreservation by optical microscopy. The classification model presented in this study can be adequate to tackle fish sperm damages.     

Structural abnormalities of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> spermatozoa

Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or “double head” in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation.