Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.     

Caspase-3在黄鳝性腺性逆转过程中的表达及定位分析

为探究黄鳝( Monopterus albus )Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝 caspase-3 的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝 caspase-3 基因cDNA序列,推导的氨基酸序列对比发现,黄鳝 caspase-3 与大黄鱼( Larimichthys crocea )和鳜( Siniperca chuatsi )亲缘关系最近。实时定量PCR ( qRT-PCR)结果显示, caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。     

Analysis of germ cell proliferation, apoptosis, and androgenesis in the Lake Van fish (Chalcalburnus tarichi) during testicular development

In the present study, the testis histology, gonadosomatic index (GSI), germ cell proliferation and apoptosis, and the plasma 11-ketotestosterone (11-KT) and testosterone (T) levels of male Chalcalburnus tarichi were analyzed. According to the histological examinations of the specimens that were caught between February 2009 and January 2010, three testicular stages were determined. Those stages were as follows: (1) recrudescence or prespawning (July–April), (2) spawning (May–June), and (3) postspawning (July). It was observed that the GSI increased gradually, starting from the recrudescence stage, and it reached peak values at the spawning stage, while the lowest values were in the postspawning. Germ cell proliferation in the testis was detected using a proliferating cell nuclear antigen (PCNA), and germ cell apoptosis was detected by transferase dUTP nick end labeling staining. The germ cell PCNA and apoptosis index values were calculated. It was indicated that germ cell proliferation was observed in all of the testicular stages. The highest germ cell PCNA index (PI) levels were detected in July, August, and September, which then dropped in October and stabilized between February and April. The lowest PI values were detected in the spawning stage (May–June). Germ cell apoptosis was observed in all of the months, and the highest apoptotic index values were detected in August, September, October, May, and June. Plasma 11-KT and T levels were at their highest levels in May and June, and it was detected as stabile in the other months. There was a correlation between GSI, PI, and plasma androgen levels. In conclusion, the present data illustrate testicular development stages for C. tarichi and show changes in the level of GSI and sex steroid biosynthesis through spermatogenesis.     

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

Development of Cre–loxP technology in zebrafish to study the regulation of fish reproduction

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.     

Increased expression of GAPDH protein is not indicative of nitrosative stress or apoptosis in liver of starved rainbow trout (Oncorhynchus mykiss)

Short-term starvation has been linked to in vivo protein degradation in liver of rainbow trout (Oncorhynchus mykiss). However, it is unclear whether this proposed increase in protein degradation is followed by programmed cell death (apoptosis) in liver of starved trout. A preliminary study in our laboratory revealed an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein that increased 4.5-fold in liver of starved trout. GAPDH is a glycolytic enzyme involved in other cellular functions, including apoptosis. Increased intracellular nitric oxide (NO) promotes nuclear translocation of GAPDH that is associated with increased apoptosis in mammals. If GAPDH protein is associated with apoptosis in rainbow trout, it could potentially be used as a biomarker of cellular stress in liver of teleost fish species. The purpose of this study was to determine whether increased GAPDH protein expression in liver of starved rainbow trout is associated with NO-induced apoptosis. Targeted proteomic analysis using multiple reaction monitoring (MRM) was used to determine the level of GAPDH in nuclear and cytoplasmic fractions and inducible nitric oxide synthase (iNOS) in cell lysates. Dot blot and DNA fragmentation analyses were conducted to evaluate protein S-nitrosylation and apoptosis, respectively. Results showed that cytoplasmic GAPDH was 3.4-fold higher in liver of starved versus fed rainbow trout but could not be detected in nuclear fractions. Starvation significantly reduced hepato-somatic index but had no effect on iNOS protein expression, protein S-nitrosylation, or apoptosis. Our results indicate that starvation promoted significant reduction in liver mass that was not associated with increased apoptosis or NO-induced stress and that greater GAPDH concentration in liver of starved rainbow trout was located primarily in the cytoplasm.     

Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer , muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.     

用流式细胞术检测喹乙醇诱导的鲤肝细胞凋亡

采用含200 mg.kg-1喹乙醇的饲料连续饲喂鲤鱼(Cyprinus carpioLinnaeus),分别于饲喂后的第7天、14天、20天、25天和第30天各剖杀4尾鱼,取肝组织,用流式细胞仪检测其细胞凋亡程度。结果显示,在肝细胞DNA直方图中,于G1峰之前出现一AP峰,且AP峰峰值随染毒时间的延长而升高,肝细胞在不同时期的凋亡率与染毒时间呈正相关。在经喹乙醇染毒第7天,鲤肝细胞的凋亡率平均值为2.05%,而对照组为1.70%,两组间差异不显著(P>0.05);在第14天,可见轻微的AP峰;在第20天,凋亡率达到9.49%,凋亡峰已较明显;在第25天时的AP峰比第14天时峰值明显升高;在染毒第30天时,凋亡率达17.44%,AP峰明显可见,其肝细胞凋亡率与第7天、第14天和第25天时差异显著。在整个过程中,对照组的正常G1峰前几乎看不出有AP峰存在。在实验组内,不同染毒时间,实验各组的肝细胞凋亡率差异显著,对照组内差异不显著。     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.     

Apoptosis does not play an important role in the resistance of 'immune' Penaeus japonicus against white spot syndrome virus

We previously demonstrated that kuruma shrimp, Penaeus japonicus, exposed to white spot syndrome virus (WSSV) became resistant ('immune' shrimp) to subsequent challenge with the virus. The present study investigated the role of apoptosis in the 'immune' shrimp during a secondary challenge with WSSV. When naive kuruma shrimp were intramuscularly injected with WSSV at a high or low dose, apoptosis was often detected by TUNEL assay in the lymphoid organ (LO), mainly in the early stage of the infection. A significantly higher incidence of apoptosis was observed in the LO of the shrimp injected with the high dose of WSSV (cumulative mortality: 100%) than in the shrimp injected with the low dose (cumulative mortality: 0%). When 'immune' and naive shrimp were injected with an equal dose of WSSV, the incidence of apoptosis was significantly lower in the 'immune' shrimp than in the naive shrimp. This difference is assumed to result from a substantial reduction of the virus by humoral neutralizing factor in the 'immune' shrimp. These results suggest that apoptosis is not a principal protective factor in 'immune' shrimp.     

低氧胁迫对鲢心肌细胞凋亡及其调控基因Bax、Bcl-2表达的影响

为了解低氧胁迫对鲢心脏的损伤及其机制,检测了不同低氧条件下鲢血清肌酸激酶(CK)活力和心肌细胞凋亡情况及凋亡调控基因Bax、Bcl-2的表达。结果显示:低氧胁迫下血清CK活性显著升高,表明低氧实验中鲢心肌细胞可能受到了损伤;TUNEL检测显示低氧胁迫鲢心肌细胞发生了凋亡,且随着氧浓度的下降凋亡指数升高,组间差异极显著;qPCR显示,鲢心肌Bax基因和Bcl-2基因变化趋势相反,Bax基因随着处理时间的延长,氧浓度的下降,表达量升高,Bcl-2基因表达量降低,且实验结束时与常氧对照值(Normoxia group,NO)比较差异显著。结果表明,低氧胁迫通过升高鲢心肌Bax基因表达、降低Bcl-2基因表达,导致心肌细胞凋亡,从而造成鲢心脏损伤甚至鱼死亡。     

温度和白消安对星点东方鲀成鱼生殖细胞枯竭的影响

为了解高温和白消安注射对星点东方鲀(Takifugu niphobles)成鱼生殖细胞发生的影响,设置32℃高温组、32℃高温-白消安组和空白对照组,比较3组的生长、存活率以及生殖细胞凋亡情况。结果显示,高温-白消安组的体重、肥满度显著低于其他组(P<0.05),而全长与其他各组差异不显著(P>0.05),各组的存活率均在90.9%以上。高温-白消安组对生殖细胞的凋亡作用比高温组更加明显,卵巢基本看不到正常的生殖细胞,且有大量的吞噬作用造成的黄褐色沉积,生殖细胞凋亡造成的空白面积比例要显著地高于高温组(P<0.05);精巢中生殖细胞凋亡的精小囊占总的精小囊的比例高达100.0%±0.0%,也显著高于高温组空泡精小囊占总的精小囊的比例(P<0.05)。得出结论:高温-白消安组较高温组更易诱导星点东方鲀内源生殖细胞凋亡,从而可制备出生殖细胞移植的适宜受体。     

黄芪多糖和当归多糖对维氏气单胞菌诱导鲫细胞凋亡的影响

为研究黄芪多糖(APS)和当归多糖(ASP)对维氏气单胞菌诱导鲫细胞凋亡的影响,实验设阴性对照组、阳性对照组,黄芪多糖组和当归多糖组,通过对鲫用维氏气单胞菌攻毒处理,用流式细胞仪测定血细胞的凋亡比例和细胞周期变化,并用荧光显微镜和透射电镜观察凋亡细胞的形态。结果显示,维氏气单胞菌攻毒后阳性对照组鲫血细胞的细胞凋亡率均极显著高于多糖组和阴性对照组;多糖组能显著降低鲫的细胞凋亡率且黄芪多糖组效果更显著;攻毒后鲫肝细胞和肾脏淋巴细胞出现染色质凝集、细胞核固缩边集和细胞空泡化及凋亡小体;同阴性对照组相比;维氏气单胞菌攻毒可以引起鲫血细胞周期中S/G2+M期细胞比例极显著下降,sub-G1极显著升高,抑制细胞分裂诱发凋亡;多糖组则G0/G1期细胞极显著降低,S/G2+M期细胞极显著升高,sub-G1极显著降低,促进细胞分裂抑制凋亡。黄芪多糖和当归多糖添加量在1%时能抑制维氏气单胞菌攻毒引起的细胞凋亡。     

A novel testicular degenerative condition in a wild population of the common carp Cyprinus carpio (L)

Gonad abnormalities can restrict or completely block reproductive capability of individuals and in some case that of their populations. Here, we describe a novel testicular degenerative condition of non-germ cell origin with a high prevalence (up to 22.1% of the population) in a wild population of carp. Based on gross morphology, and microscopic and cellular examinations, the condition shows progressive severity which could be categorized into low, mild, severe and complete. In early stages of the condition, an abnormally increased proliferation (11-fold) of the Sertoli cell occurred, followed by degenerative cell death of all testicular cells, resulting in fluid-filled vesicles in the later stages. This initial uncontrolled proliferation of Sertoli cells suggests that the condition could be triggered by malignant pathways; however, the observed subsequent apoptosis of all testicular cells en masse, rendering the animals “sterile,” appears unique. Observations, to date, indicate that this condition is specific to male carp and not present in other species of fish sharing the habitat. High prevalence of the condition allowed comparative evaluation between affected individuals, an aspect likely to facilitate future studies, including elucidation of the cause, robust testing of therapies and practical applications such as management of feral carp populations.     

Gonadal histology and some biochemical characteristics of <Emphasis Type="Italic">Chalcalburnus tarichi</Emphasis> (Pallas, 1811) having abnormal gonads

The gonad histology, gonado-somatic index (GSI), 17β-estradiol (E₂) levels and acetylcholinesterase (AChE) activity in the carp species Chalcalburnus tarichi from Lake Van and the Karasu river, eastern Turkey, have been investigated. Fish between 5 and 7 years old were sampled from November 2003 to February 2004. The ratio of female fish caught in Lake Van with abnormal ovaries (AbOF) was 43.3%, but the fork length and body weight of these fish were not correlated with this abnormality. The weight of the ovaries and the GSI values of AbOF were very low (P < 0.05). Histological observations on the samples caught each month revealed that the oocytes had degenerated in the perinucleolus and early cortical alveolus stages and that the ovaries were full of somatic stromal tissue. In addition, the seminiferous tubules of male fish with abnormal testes did not contain male reproductive cells at any stage. The ovaries of the fish caught from the Karasu river were also full of oocytes in the perinucleolus and early cortical alveolus stages, but there were fewer atretic follicles. Furthermore, apoptosis was observed in the ovary cells of these fish, in particular in the follicular cells, and the plasma E₂ levels of the AbOF was very low (P < 0.05). AChE activity was inhibited only in liver (P < 0.05). We conclude that our sample of C. tarichi must have been exposed to various polluting chemicals or another unknown factors (such as global warming) and that these factors have irreversibly impaired oocyte development in a high percentage of fish.     

Apoptosis in Ichthyophthirius multifiliis is associated with expression of the Fas receptor of theronts

The expression of type I membrane Fas receptors on the surface of Ichthyophthirius multifiliis (Ich) theronts and the possible association between Fas expression and theront apoptosis induced by the immune antibody was examined. Fas receptors were detected on the theront surface using fluorescein isothiocyanate-conjugated mouse monoclonal antibody against Fas. Fas-positive theronts significantly increased with time during in vitro incubation and with increasing theront concentration. Furthermore, the immune cutaneous antibody induced theront apoptosis; however, Fas ligand did not. A highly significant correlation was noted between theront Fas expression and immune cutaneous antibody-induced theront apoptosis. Numbers of apoptotic theronts increased with increasing number of Fas-positive theronts. The data indicated that theront apoptosis induced by immune cutaneous antibody appears to be positively correlated with the expression of Fas on the surface of Ich theronts.     

遗传全雄尼罗罗非鱼与吉富罗非鱼养殖性能比较

以超雄鱼为父本进行遗传全雄鱼的繁育是获得全雄罗非鱼苗种的方法之一。为了评估全雄尼罗罗非鱼的养殖性能,进行了遗传全雄尼罗罗非鱼与吉富罗非鱼的池塘及网箱养殖比较试验。结果显示,初始体长3～5 cm的遗传全雄尼罗罗非鱼和吉富罗非鱼鱼苗经过100 d池塘养殖后,平均体质量分别达到558.00 g和501.80 g,遗传全雄群体的生长速度比吉富群体快11.26%;将体质量为18～19 g的遗传全雄尼罗罗非鱼和吉富罗非鱼鱼种放在同一网箱中饲养,45 d后,其平均体质量分别达到144.69 g和125.91 g,遗传全雄群体的生长速度比吉富群体快18.02%;同时,遗传全雄群体的雄性率达到了100%。对比试验结果表明,遗传全雄尼罗罗非鱼具有较高的养殖推广价值。     

Effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio in vivo

To investigate the effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio in vivo, the fish were exposed to 0, 0.5, 1.0, 2.0 and 4.0?mg?l(-1) trichlorfon concentrations for 30?days. At the end of the experiment, plasm antioxidant activities, hepatic total nitric oxide synthase (T-NOS) activity, xanthine oxidase (XOD) activity and hepatocyte apoptosis rate were analyzed. The results showed hepatic T-NOS activities, XOD activities and hepatocyte apoptosis rate were increased with trichlorfon concentration increase. The hepatic MDA content was increased significantly at 4.0?mg?l(-1) trichlorfon treatment. The disturbance of antioxidative balance was observed based on the monitoring of the plasm superoxide dismutase (SOD), catalase (CAT) activities and vitamin E levels. The current study revealed that trichlorfon influenced fish plasma antioxidative status, caused lipid peroxidation (LPO) then resulted in hepatocytes apoptosis.     

Induction of apoptosis in tilapia, Oreochromis aureus Steindachner, and in TO-2 cells by Staphylococcus epidermidis

This study describes apoptosis induced in vivo and in vitro in tilapia, Oreochromis aureus Steindachner, by Staphylococcus epidermidis . In an in vivo experiment, tilapia were challenged using viable S. epidermidis and its cultured supernatant, respectively. Apoptosis was predominantly detected in lymphocytes and macrophages in spleen and kidney. Apoptotic figures were observed occasionally in the brain, liver, gonad, mesentery, stomach, intestine and skeletal muscle of infected fish. In an in vitro experiment, TO-2 cells treated with brain heart infusion broth supernatant of the bacteria after 48 h revealed that the supernatant was able to induce the cell apoptosis. Fragmented DNA was also detected 48 h after treatment using 1.5% agarose gel electrophoresis. The smallest multimeric DNA fragment was approximately 180-bp in length. Results of both the in vitro and in vivo experiments showed that products of the bacteria could induce tilapia cell apoptosis.     

Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii

Haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, were investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated haemocytes of M. rosenbergii were cultured at a ratio of 1:50 haemocytes to pathogen with the yeast Debaryomyces hansenii, the bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25 degrees C for 2 h, followed by washing to remove free particles. At least 200 haemocytes were counted to determine the phagocytosis rate, and the results showed that haemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing haemocytes displayed morphological changes characteristic of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation and fragmentation of nuclei. This pathogen-induced apoptosis was further confirmed by DNA laddering and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labelling) assays. Neither haemocytes treated with latex beads nor uninfected haemocytes (control group) showed signs of apoptosis after 48 h in culture.