Pitts Natalie L, Mykles Donald L
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
Comp Biochem Physiol A Mol Integr Physiol. 2017 Jan;203:328-340. doi: 10.1016/j.cbpa.2016.10.012. Epub 2016 Oct 27.
In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~10-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iβ) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.
在十足目甲壳动物中,蜕皮受眼柄神经节(ESG)中X器官/窦腺(XO/SG)复合体神经分泌细胞脉冲式释放蜕皮抑制激素(MIH)的控制。MIH释放量的下降通过激活蜕皮腺或Y器官(YO)来触发蜕皮。转录后机制最终控制血淋巴中MIH的水平。神经递质介导的电活动控制MIH从窦腺轴突末端的钙依赖性囊泡释放,这可能受到一氧化氮(NO)的调节。在绿岸蟹Carcinus maenas中,窦腺中存在一氧化氮合酶(NOS)蛋白和NO。此外,C. maenas对眼柄切除(ESA)具有抗性,这表明神经系统的其他区域分泌足够量的MIH以防止蜕皮。相比之下,ESA可诱导黑背陆蟹Gecarcinus lateralis蜕皮。采用双标免疫荧光显微镜和定量聚合酶链反应分别对C. maenas和G. lateralis的ESG、脑和胸神经节(TG)中的MIH和NOS蛋白及转录本进行定位和定量。在ESG中,两种物种的窦腺中MIH免疫阳性细胞和NOS免疫阳性细胞紧密相连;共聚焦显微镜显示NOS定位于与MIH阳性轴突末端相邻的细胞中。在脑中,MIH阳性细胞位于嗅叶中的少数细胞中;未检测到NOS免疫荧光。在TG中,MIH和NOS定位于节段神经之间的细胞簇中。在G. lateralis中,蜕皮间期动物的ESG中Gl-MIH和Gl-甲壳动物高血糖激素(CHH)mRNA水平比脑或TG中的高约10倍,表明ESG是这些神经肽的主要来源。ESG中Gl-NOS和Gl-延伸因子(EF2)mRNA水平也较高。蜕皮阶段对两种物种ESG中的CHH、NOS、NOS相互作用蛋白(NOS-IP)、膜鸟苷酸环化酶-II(GC-II)和非NO依赖性GC-III表达几乎没有影响。相比之下,G. lateralis在蜕皮前和蜕皮后阶段MIH和NO受体GC-Iβ亚基(GC-Iβ)转录本增加,而C. maenas则没有。脑和TG中的MIH免疫阳性细胞可能是MIH的次要来源;这些来源释放的MIH可能导致两种物种对ESA反应的差异。TG中的MIH免疫阳性细胞可能是介导肢体芽自切蜕皮抑制的MIH样因子的来源。ESG和TG中MIH和NOS标记细胞的关联表明NO可能调节MIH释放。提出了一个模型,其中GC-I的NO依赖性激活抑制MIH囊泡与神经末梢膜的钙依赖性融合;MIH的减少激活YO,动物进入蜕皮前阶段。