Dataset Information

KIFC1 is essential for normal spermatogenesis and its depletion results in early germ cell apoptosis in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus.

ABSTRACT: In order to explore the dynamic mechanisms during spermatogenesis of the penaeid prawns, the full length of kifc1 was cloned from testis cDNA of Penaeus japonicus through RACE. Both semi-quantitative RT-PCR and Western blot results indicated that KIFC1 was extensive expressed in different tissue of P. japonicus. Compared with other tissue, the highest expression of KIFC1 occurred in the testis. According to the immunofluorescence results, the KIFC1 protein was detected at each stage of whole process of spermatogenesis. In the spermatogonial phase, KIFC1 mainly dispersed in cytoplasm and co-localized with microtubules, while abundant KIFC1 signal was detected in the nucleus of spermatocytes. At the early stage of spermatids, KIFC1 was transported from the nucleus into the cytoplasm, and it assisted microtubule assembly onto one side of the nucleus. Finally, in mature sperm, it was weakly expressed in the acrosome. This implies that KIFC1 may participate in the mitosis of spermatogonia, meiosis of spermatocyte, and acrosome formation during spermiogenesis; it may also play functions in acrosome maintaining in mature sperm. In addition, the results of KIFC1 knockdown by dsRNA injection in vivo reveal that decreased KIFC1 expression may induce aberrant microtubule assembly, and it leads to spermatogonia and spermatocyte apoptosis.

SUBMITTER: Hao SL

PROVIDER: S-EPMC6949060 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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KIFC1 is essential for normal spermatogenesis and its depletion results in early germ cell apoptosis in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus.

Hao Shuang-Li SL Yang Wan-Xi WX

Aging 20191229 24

In order to explore the dynamic mechanisms during spermatogenesis of the penaeid prawns, the full length of kifc1 was cloned from testis cDNA of Penaeus japonicus through RACE. Both semi-quantitative RT-PCR and Western blot results indicated that KIFC1 was extensive expressed in different tissue of P. japonicus. Compared with other tissue, the highest expression of KIFC1 occurred in the testis. According to the immunofluorescence results, the KIFC1 protein was detected at ea ...[more]

PMID: 31895691

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Project description:BACKGROUND: Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome. RESULTS: Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences. CONCLUSIONS: Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.

Project description:Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -β, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.

Project description:BackgroundAcrosome formation and nuclear shaping are the main events in spermatogenesis. During spermiogenesis in Exopalaemon modestus, a unique microtubular structure called the acroframosome (AFS) forms in spermatids. The AFS links to a temporary organelle called the lamellar complex (LCx) leading to the formation of an everted umbrella-shaped acrosome and a dish-shaped nucleus in the mature sperm. These morphological changes require complex cell motility in which the C-terminal kinesin motor protein called KIFC1 is involved. In this study, we demonstrate that KIFC1 moves along the AFS and plays an important role in acrosome formation and nuclear shaping during spermatogenesis in E. modestus.Methodology/principal findingsWe cloned a 3125 bp complete cDNA of kifc1 from the testis of E. modestus by PCR. The predicted secondary and tertiary structures of E. modestus KIFC1 contain three domains: a) the C-terminus, b) the stalk region, and the c) N-terminusl. Semi-quantitative RT-PCR detected the expression of kifc1 mRNA in different tissues of E. modestus. In situ hybridization demonstrated the temporal and spatial expression profile of kifc1 during spermiogenesis. Western blot identified the expression of KIFC1 in different tissues of E. modestus, including the testis. Immunofluorescence localized KIFC1, tubulin, GM130, and mitochondria in order to elucidate their role during spermiogenesis in E. modestus.Conclusion/significanceOur results indicate that KIFC1 transports the Golgi complex, mitochondria, and other cellular components that results in acrosome formation and nuclear shaping in E. modestus. The KIFC1 transport function depends upon the microtubular structure called the acroframosome (AFS). This study describes some of the molecular mechanisms involved in the acrosome formation and nuclear shaping in E. modestus. In addition, this study may provide a model for studying the molecular mechanisms involved in spermatogenesis in other crustacean species and lead to a better understanding of the fertilization process in crustaceans.

Dataset Information

KIFC1 is essential for normal spermatogenesis and its depletion results in early germ cell apoptosis in the Kuruma shrimp, Penaeus (Marsupenaeus) japonicus.

Publications

KIFC1 is essential for normal spermatogenesis and its depletion results in early germ cell apoptosis in the Kuruma shrimp, <i>Penaeus (Marsupenaeus) japonicus</i>.

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