Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus

Abstract

Eukaryotic elongation factor 1 alpha (eEF1A) is an essential component of the translational apparatus. In the present study, eEF1A1b was isolated from the Nile tilapia. Real-time PCR and Western blot revealed that eEF1A1b was expressed highly in the testis from 90 dah (days after hatching) onwards. In situ hybridization and immunohistochemistry analyses showed that eEF1A1b was highly expressed in the spermatogonia of the testis. CRISPR/Cas9 mediated mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in the F0 XY fish. Consistently, heterozygous mutation of eEF1A1b (eEF1A1b+/−) resulted in an absence of spermatocytes at 90 dah, very few spermatocytes, spermatids and spermatozoa at 180 dah, and decreased Cyp11b2 and serum 11-ketotestosterone level at both stages. Further examination of the fertilization capacity of the sperm indicated that the eEF1A1b+/− XY fish were infertile due to abnormal spermiogenesis. Transcriptomic analyses of the eEF1A1b+/− testis from 180 dah XY fish revealed that key elements involved in spermatogenesis, steroidogenesis and sperm motility were significantly down-regulated compared with the control XY. Transgenic overexpression of eEF1A1b rescued the spermatogenesis arrest phenotype of the eEF1A1b+/− testis. Taken together, our data suggested that eEF1A1b is crucial for spermatogenesis and male fertility in the Nile tilapia.

Genome-Wide Identification, Evolution and Expression of the Complete Set of Cytoplasmic Ribosomal Protein Genes in Nile Tilapia

“…Furthermore, RPL7-1, RPL8, RPL11, RPL14, RPL18, RPL23A, RPL24, RPS5 and RPS24 may play important roles in the development of testis and spermatogenesis, while RPL5b and RPS27-1b may play important roles in the development of ovary and oogenesis. Sex-specific expression of translation elongation factors eEF1α1b in the testis and 42Sp50 in the ovary have been reported in frogs and several fish species, including tilapia [51][52][53][54]. Taken together, these results indicate that the gonad tightly controls its biosynthesis machinery to meet the needs of proteins required for oogenesis and spermatogenesis.…”

Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay

“…However, gene EEF1A1 , whose super enhancer was found to be highly correlated with SLC17A5 (both genes located in the same topological domain, Fig. 4B), 31 was likely haploinsufficient and might be essential for spermatogenesis 37 . It indicated that dysregulation of EEF1A1 caused by disruption of SLC17A5 would be the potential pathogenicity.…”

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

“…In tilapia, varied concentrations of gRNAs (50–250 ng/μL) have been used to date 15 , 21 , 22 , but there is a lack of data on the optimal ratio of sgRNA to Cas9 to ensure high mutagenesis efficiency and low treatment mortality. We tested three different concentrations of sgRNA (100, 150 and 250 ng/µL) with a constant concentration of Cas9 mRNA (500 ng/µL), with molar ratios of Cas9:sgRNA being 1:9.0, 1:13.4 and 1:22.4, respectively.…”

“…The lack of understanding of the resultant mutations including the level of mosaicism and the indel spectrum hinders the direct functional analysis in the injected animals. The most frequently used screening methods in gonad-related gene functional studies have been restriction enzyme digestion (RED) and Sanger sequencing of a limited number of cloned sequences [8][9][10][11][12][13]15,[21][22][23] , with a few studies adopting high resolution melt curve analysis (HRMA) or SURVEYOR techniques 24,25 . Such approaches have a number of potential limitations in their accuracy to describe KO effects on target genotypes, which are further confounded by the pooling of samples which ultimately provides a biased interpretation of the efficacy of CRISPR/Cas9 gene editing in this field.…”