MiR-26 regulates ddx3x expression in medaka (Oryzias latipes) gonads.

MicroRNAs (miRNAs) participate in the reproductive process by regulating the expression of target genes, however, there are few studies on the regulation of mRNA target by miRNAs in medaka (Oryzias latipes). Our previous data showed that miR-202-5p has a sex-biased expression and miR-26 is abundantly expressed in both ovary and testis of medaka. In this study, DEAD-box helicase 3, X-linked (ddx3x) was proposed to be a putative target of miR-26 and miR-202-5p through bioinformatic analysis. Here, medaka ddx3x (Olddx3x) was cloned and sequence alignment analysis indicated that Olddx3x is highly conserved and has more than 90% identity with some other teleosts. Reverse transcription polymerase chain reaction (RT-PCR) showed that Olddx3x was highly expressed in testis and ovary. Meanwhile, chromogenic in situ hybridization manifested that Olddx3x was abundantly expressed in the early oocytes in the ovary and in various stages of spermatogenesis, especially the obvious signal in the testis of spermatocytes and sperm. Dual-luciferase reporter assay and the injection of miR-26 agomir and antagomir demonstrated that the expression of Olddx3x was regulated by miR-26 but not miR-202-5p. The overall results revealed that miR-26 may perform a vital role in medaka reproduction development by regulating the expression of ddx3x.

In vivo injection of α-bungarotoxin to improve the efficiency of motor endplate labeling.

INTRODUCTION: Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent α-bungarotoxin. METHODS: Fifty-two mice were randomly divided into two groups, an experiment group (n = 50), and a contrast group (n = 2). In experiment group, α-bungarotoxin was injected via the caudal vein. The injection dosages were designated as 0.1, 0.2, 0.3, 0.4, and 0.5 µg/g. The experimental mice were divided into five subgroups of ten mice per group. The contrast group was only injected with 200 µL normal saline solution. Bilateral gastrocnemius were acquired for microscope analysis and optical clearing to seek specific fluorescent signal. RESULTS: A dose of 0.3 µg/g of α-bungarotoxin with 1 h conjugation time could display the number and structure of motor endplate in plane view. Compared with the traditional procedure, this method was rapid, convenient, and time-saving. Combined with the optical clearing technique, spatial distribution could also be seen, helping to better understand the stereoscopic view of motor endplate position in skeletal muscle. CONCLUSIONS: In vivo injection of α-bungarotoxin proved effective for studying motor endplate in skeletal muscle.