The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Rbm46, a novel germ cell-specific factor, modulates meiotic progression and spermatogenesis.

It has been suggested that many novel RNA-binding proteins (RBPs) are required for gametogenesis, but the necessity of few of these proteins has been functionally verified. Here, we identified one RBP, Rbm46, and investigated its expression pattern and role in zebrafish reproduction. We found that rbm46 is maternally provided and specifically expressed in the germ cells of gonadal tissues using in situ hybridization, reverse transcription-PCR, and quantitative real-time polymerase chain reaction (qRT-PCR). Two independent rbm46 mutant zebrafish lines were generated via the transcription activator-like effector nuclease technique. Specific disruption of rbm46 resulted in masculinization and infertility in the mutants. Although the spermatogonia appeared grossly normal in the mutants, spermatogenesis was impaired, and meiosis events were not observed. The introduction of a tp53M214K mutation could not rescue the female-to-male sex-reversal phenotype, indicating that rbm46 acts independently of the p53-dependent apoptotic pathway. RNA sequencing and qRT-PCR subsequently indicated that Rbm46 might be involved in the posttranscriptional regulation of functional genes essential for germ cell development, such as nanos3, dazl, and sycp3, during gametogenesis. Together, our results reveal for the first time the crucial role of rbm46 in regulating germ cell development in vivo through promotion of germ cell progression through meiosis prophase I.

Emergence of breeding tubercles and puberty onset in male zebrafish: evidence for a dependence on body growth.

The presence of breeding tubercles (BTs) on the pectoral fins has been investigated as a typical male secondary sexual characteristic (SSC) that distinguish males from females in adult zebrafish. Nonetheless, the earliest occurrence of these tubercles and its association with puberty onset and body growth remain unclear. In this study, using morphological, histological and statistical analyses, the authors examined the first appearance of BTs and puberty onset in male zebrafish, with particular emphasis on the potential impact of body growth on them. The results of this study revealed that BTs distributed along the first five branched pectoral fin rays were the earliest manifestation of male SSCs, which is significantly strongly correlated with body weight (R2  = 0.9609, P < 0.001), and could be used as a "gold standard" for the earliest sex distinction (<0.1 g in weight). Using the first appearance of BTs (<0.20 mm2 ) as a metric, the authors established that male puberty commenced at a body weight of c. 0.056 ± 0.015 g or a standard length of 10.99 ± 1.051 mm (mean ± S.D.). In this study, the authors thus established a simple method that can be used to sex live zebrafish at the pubertal stage and provides the first evidence for the relationship of BTs and male puberty initiation with body growth. These findings will accordingly lay a foundation for exploring mechanisms of the SSCs and male puberty onset in zebrafish and other teleost fish.

Adenine nucleotide translocase 2 (Ant2) is required for individualization of spermatogenesis of Drosophila melanogaster.

Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.

RpL38 modulates germ cell differentiation by controlling Bam expression in Drosophila testis.

Switching from mitotic spermatogonia to meiotic spermatocytes is critical to producing haploid sperms during male germ cell differentiation. However, the underlying mechanisms of this switch remain largely unexplored. In Drosophila melanogaster, the gene RpL38 encodes the ribosomal protein L38, one component of the 60S subunit of ribosomes. We found that its depletion in spermatogonia severely diminished the production of mature sperms and thus led to the infertility of male flies. By examining the germ cell differentiation in testes, we found that RpL38-knockdown blocked the transition from spermatogonia to spermatocytes and accumulated spermatogonia in the testis. To understand the intrinsic reason for this blockage, we conducted proteomic analysis for these spermatogonia populations. Differing from the control spermatogonia, the accumulated spermatogonia in RpL38-knockdown testes already expressed many spermatocyte markers but lacked many meiosis-related proteins, suggesting that spermatogonia need to prepare some important proteins for meiosis to complete their switch into spermatocytes. Mechanistically, we found that the expression of bag of marbles (bam), a crucial determinant in the transition from spermatogonia to spermatocytes, was inhibited at both the mRNA and protein levels upon RpL38 depletion. We also confirmed that the bam loss phenocopied RpL38 RNAi in the testis phenotype and transcriptomic profiling. Strikingly, overexpressing bam was able to fully rescue the testis abnormality and infertility of RpL38-knockdown flies, indicating that bam is the key effector downstream of RpL38 to regulate spermatogonia differentiation. Overall, our data suggested that germ cells start to prepare meiosis-related proteins as early as the spermatogonial stage, and RpL38 in spermatogonia is required to regulate their transition toward spermatocytes in a bam-dependent manner, providing new knowledge for our understanding of the transition process from spermatogonia to spermatocytes in Drosophila spermatogenesis.