scholarly journals Potential Functions of Gem-Associated Protein 2-Like Isoform X1 in the Oriental River Prawn Macrobrachium nipponense: Cloning, qPCR, In Situ Hybridization, and RNAi Analysis

2019 ◽  
Vol 20 (16) ◽  
pp. 3995 ◽  
Author(s):  
Shubo Jin ◽  
Yuning Hu ◽  
Hongtuo Fu ◽  
Sufei Jiang ◽  
Yiwei Xiong ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sexual Development ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Negative Effects ◽  
Macrobrachium Nipponense ◽  
Reading Frame ◽  
Gonad Differentiation ◽  
Qpcr Analysis

Gem-associated protein 2-like isoform X1 (GEM) was previously predicted to be involved in the sexual development of male Macrobrachium nipponense. In this study, we analyze the GEM functions in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, and RNA interference (RNAi). The full-length Mn-GEM cDNA sequence was 1018 base pairs (bp) long with an open reading frame of 777 bp encoding 258 amino acids. qPCR analysis of Mn-GEM in different tissues and developmental stages showed that Mn-GEM was highly expressed in the gonad and from post-larval developmental stage day 5 (PL5) to PL15, which indicated that GEM has potential roles in gonad differentiation and development in M. nipponense. In situ hybridization and qPCR analysis of various stages of the reproductive cycle of the testis and ovary indicated that GEM may promote spermatid development and gametogenesis in M. nipponense. After injecting with double-stranded RNA (dsRNA) of Mn-GEM, mRNA expression of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone increased with the decrease of Mn-GEM expression, indicating that GEM has negative effects on the male sexual differentiation and development in M. nipponense. Results of this study highlight the functions of GEM in M. nipponense, which can be applied to future studies of male sexual development in M. nipponense and other crustacean species.

scholarly journals Identification and Characterization of the Pyruvate Dehydrogenase E1 Gene in the Oriental River Prawn, Macrobrachium nipponense

2021 ◽  
Vol 12 ◽  
Author(s):  
Shubo Jin ◽  
Yuning Hu ◽  
Hongtuo Fu ◽  
Sufei Jiang ◽  
Yiwei Xiong ◽  
...  
Keyword(s):  
Sexual Development ◽  
Pyruvate Dehydrogenase ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Types ◽  
Macrobrachium Nipponense ◽  
Reading Frame ◽  
Testis Development ◽  
Positive Effects ◽  
Oriental River Prawn

Pyruvate dehydrogenase E1 (PDHE1) is thought to play essential roles in energy metabolism, and a previous study suggested that it also has potential regulatory roles in male sexual development in the oriental river prawn, Macrobrachium nipponense. In this study, we used rapid amplification of cDNA ends, quantitative polymerase chain reaction (qPCR), in situ hybridization, western blotting, RNA interference (RNAi), and histological analyses to assess the potential functions of Mn-PDHE1 in the sexual development of male M. nipponense. The full cDNA sequence of Mn-PDHE1 was 1,614 base pairs long, including a 1,077 base pair open reading frame that encodes 358 amino acids. qPCR analysis revealed the regulatory functions of PDHE1 in male sexual development in M. nipponense and in the metamorphosis process. In situ hybridization and western blot results indicated that PDHE1 was involved in testis development, and RNAi analysis showed that PDHE1 positively regulated the expression of insulin-like androgenic gland factor in M. nipponense. Compared with the cell types in the testes of control prawns, histological analysis showed that the number of sperm was dramatically lower after test subjects were injected with Mn-PDHE1 dsRNA, whereas the numbers of spermatogonia and spermatocytes were higher. Sperm constituted only 1% of cells at 14 days after injection in the RNAi group. This indicated that knockdown of the expression of PDHE1 delayed testis development. Thus, PDHE1 has positive effects on male sexual development in M. nipponense. This study highlights the functions of PDHE1 in M. nipponense and its essential roles in the regulation of testis development.

scholarly journals Identification and Characterization of the Succinate Dehydrogenase Complex Iron Sulfur Subunit B Gene in the Oriental River Prawn, Macrobrachium nipponense

2021 ◽  
Vol 12 ◽  
Author(s):  
Shubo Jin ◽  
Yuning Hu ◽  
Hongtuo Fu ◽  
Sufei Jiang ◽  
Yiwei Xiong ◽  
...  
Keyword(s):  
Western Blot ◽  
Sexual Development ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Full Genome Sequence ◽  
Macrobrachium Nipponense ◽  
Testis Development ◽  
Positive Effects ◽  
Dsrna Injection

Previous studies have revealed that SDHB has potential functions in the male sexual differentiation and development in M. nipponense through providing ATP. In this study, the functions of Mn-SDHB were further analyzed in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, western-blot, and RNA interference (RNAi), combined with the histological observations. The full-genome sequence of Mn-SDHB was 54,608 bp at Chromosome 34, including 7 introns and 6 exons. The full-length cDNA sequence of Mn-SDHB was 1,268 base pairs (bp) long with an open reading frame of 807 bp, encoding for 268 amino acids. The highest expression level of Mn-SDHB in different tissues was observed in the testis, and male prawns at post-larval developmental stage 25 during different developmental stages, indicating that SDHB was potentially involved in the male sexual development in M. nipponense. In situ hybridization and western-blot analysis indicated that SDHB plays essential roles in the testis development. The in situ hybridization analysis also implies the potential roles of Mn-SDHB in ovarian development. The expressions of Mn-IAG were decreased after Mn-SDHB dsRNA injection, indicating SDHB has the positive regulatory effects on IAG in M. nipponese. Thus, SDHB was involved in the mechanism of the male sexual development. The testis development was inhibited, and sperms were rarely observed after 10 days of Mn-SDHB dsRNA injection, indicating SDHB has positive effects on the male sexual development in M. nipponense. This study highlights the functions of SDHB in M. nipponense, which provide new insights for the future studies of the male sexual development in other crustacean species.

scholarly journals Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

2022 ◽  
Vol 12 ◽  
Author(s):  
Sufei Jiang ◽  
Yiwei Xiong ◽  
Wenyi Zhang ◽  
Junpeng Zhu ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Cysteine Proteases ◽  
Macrobrachium Nipponense ◽  
Reading Frame ◽  
Physiological Processes ◽  
Gonad Somatic Index ◽  
Oriental River Prawn ◽  
Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 77-85 ◽  
Author(s):  
M.L. Snead ◽  
W. Luo ◽  
E.C. Lau ◽  
H.C. Slavkin
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Gene Transcription ◽  
Developmental Stages ◽  
Three Dimensional ◽  
Molar Tooth ◽  
Specific Expression ◽  
Amelogenin Gene ◽  
Stage Of Development

Position- and time-restricted amelogenin gene transcription was analysed in developing tooth organs using in situ hybridization with asymmetric complementary RNA probes produced from a cDNA specific to the mouse 26 × 10(3) Mr amelogenin. In situ analysis was performed on developmentally staged fetal and neonatal mouse mandibular first (M1) and maxillary first (M1) molar tooth organs using serial sections and three-dimensional reconstruction. Amelogenin mRNA was first detected in a cluster of ameloblasts along one cusp of the M1 molar at the newborn stage of development. In subsequent developmental stages, amelogenin transcripts were detected within foci of ameloblasts lining each of the five cusps comprising the molar crown form. The number of amelogenin transcripts appeared to be position-dependent, being more abundant on one cusp surface while reduced along the opposite surface. Amelogenin gene transcription was found to be bilaterally symmetric between the developing right and left M1 molars, and complementary between the M1 and M1 developing molars; indicating position-restricted gene expression resulting in organ stereoisomerism. The application of in situ hybridization to forming tooth organ geometry provides a novel strategy to define epithelial-mesenchymal signal(s) which are believed to be responsible for organ morphogenesis, as well as for temporal- and spatial-restricted tissue-specific expression of enamel extracellular matrix.

scholarly journals Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense)

2019 ◽  
Vol 20 (8) ◽  
pp. 1856 ◽  
Author(s):  
Shengming Sun ◽  
Ying Wu ◽  
Hongtuo Fu ◽  
Xianping Ge ◽  
Hongzheng You ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Open Reading Frames ◽  
Dependent Manner ◽  
Macrobrachium Nipponense ◽  
Oriental River Prawn ◽  
Adverse Environmental Conditions ◽  
The Brain ◽  
Identification And Characterization

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.

scholarly journals Type IV collagen mRNA accumulates in the mesenchymal compartment at early stages of murine developing intestine.

1990 ◽  
Vol 110 (3) ◽  
pp. 849-857 ◽  
Author(s):  
P Simon-Assmann ◽  
F Bouziges ◽  
J N Freund ◽  
F Perrin-Schmitt ◽  
M Kedinger
Keyword(s):  
In Situ Hybridization ◽  
Type Iv Collagen ◽  
Developmental Stages ◽  
Cdna Probe ◽  
Mesenchymal Cells ◽  
Hybridization Signal ◽  
Collagen Mrna ◽  
Early Stages ◽  
Type Iv

The expression of type IV collagen mRNA during mouse intestinal morphogenesis was examined by in situ hybridization using a cDNA probe corresponding to mRNA for alpha 1 (IV) chain. Type IV collagen mRNA is detected in the embryonic mesenchymal cells at early stages of development (12 d of gestation). A segregation of mesenchymal cells expressing high levels of type IV collagen mRNA in close vicinity of the epithelium occurs just before villus formation. During villus outgrowth, type IV collagen mRNA, still confined to mesenchyme-derived tissues, is progressively restricted to the mucosal connective tissue (the lamina propria) and to a lesser extent to the muscular layers. In the adult, the amount of messenger is quite low as compared to the level found in the developing intestine and the in situ hybridization signal, indistinguishable from the background, is uniform throughout the whole intestinal wall. At all developmental stages no detectable specific hybridization signal is virtually observed over the epithelium cell layer. These results show that high amounts of the type IV collagen messenger are detected during phases of intensive morphogenetic events. Furthermore, they reinforce the notion already gained previously (Simon-Assmann et al. 1988) that the mesenchymal compartment is the principal endogenous source of type IV collagen. They also indicate that the continuous migration of epithelial cells along the basement membrane of intestinal villi in the mature organ is not accompanied by a significant remodeling of the collagen IV network.

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