Dataset Information

Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

ABSTRACT: Individual sperm cells are resolved from a syncytium during late step of spermiogenesis known as individualization, which is accomplished by an Individualization Complex (IC) composed of 64 investment cones. mulet encodes Tubulin-binding cofactor E-like (TBCEL), suggesting a role for microtubule dynamics in individualization. Indeed, a population of ?100 cytoplasmic microtubules fails to disappear in mulet mutant testes during spermatogenesis. This persistence, detected using epi-fluorescence and electron microscopy, suggests that removal of these microtubules by TBCEL is a prerequisite for individualization. Immunofluorescence reveals TBCEL expression in elongated spermatid cysts. In addition, testes from mulet mutant males were rescued to wild type using tubulin-Gal4 to drive TBCEL expression, indicating that the mutant phenotype is caused by the lack of TBCEL. Finally, RNAi driven by bam-GAL4 successfully phenocopied mulet, confirming that mulet is required in the germline for individualization. We propose a model in which the cytoplasmic microtubules serve as alternate tracks for investment cones in mulet mutant testes.This article has an associated First Person interview with the first author of the paper.

SUBMITTER: Fabrizio JJ

PROVIDER: S-EPMC7055396 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

Fabrizio James J JJ Rollins Janet J Bazinet Christopher W CW Wegener Stephanie S Koziy Iryna I Daniel Rachel R Lombardo Vincent V Pryce Dwaine D Bharrat Kavita K Innabi Elissa E Villanobos Marielle M Mendoza Gabriela G Ferrara Elisa E Rodway Stephanie S Vicioso Matthew M Siracusa Victoria V Dailey Erin E Pronovost Justin J Innabi Simon S Patel Vrutant V DeSouza Nicole N Quaranto Danielle D Niknejad Amir A

Biology open 20200226 2

Individual sperm cells are resolved from a syncytium during late step of spermiogenesis known as individualization, which is accomplished by an Individualization Complex (IC) composed of 64 investment cones. mulet encodes Tubulin-binding cofactor E-like (TBCEL), suggesting a role for microtubule dynamics in individualization. Indeed, a population of ∼100 cytoplasmic microtubules fails to disappear in mulet mutant testes during spermatogenesis. This persistence, detected using epi-f ...[more]

PMID: 32033965

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Project description:Spermatogenesis in all animal species occurs within a syncytium. Only at the very end of spermatogenesis are individual sperm cells resolved from this syncytium in a process known as individualization. Individualization in Drosophila begins as a membrane-cytoskeletal complex known as the individualization complex (IC) assembles around the sperm heads and proceeds down the flagella, removing cytoplasm from between the sperm tails and shrink-wrapping each spermatid into its own plasma membrane as it travels. The mulet (mlt) mutation results in severely disrupted ICs, indicating that the mlt gene product is required for individualization. Inverse PCR followed by cycle sequencing maps all known P-insertion alleles of mlt to two overlapping genes, CG12214 (the Drosophila tubulin-binding cofactor E-like homolog) and KCNQ (a large voltage-gated potassium channel). However, since the alleles of mlt map to the 5'-UTR of CG12214 and since CG12214 is contained within an intron of KCNQ, it was hypothesized that mlt and CG12214 are allelic. Indeed, CG12214 mutant testes exhibited severely disrupted ICs and were indistinguishable from mlt mutant testes, thus further suggesting allelism. To test this hypothesis, alleles of mlt were crossed to CG12214 in order to generate trans-heterozygous males. Testes from all trans-heterozygous combinations revealed severely disrupted ICs and were also indistinguishable from mlt mutant testes, indicating that mlt and CG12214 fail to complement one another and are thus allelic. In addition, complementation testing against null alleles of KCNQ verified that the observed individualization defect is not caused by a disruption of KCNQ. Finally, since a population of spermatid-associated microtubules known to disappear prior to movement of the IC abnormally persists during individualization in CG12214 mutant testes, this work implicates TBCE-like in the removal of these microtubules prior to IC movement. Taken together, these results identify mlt as CG12214 and suggest that the removal of microtubules by TBCE-like is a necessary pre-requisite for proper coordinated movement of the IC.

Project description:Spermiogenesis is the longest phase of spermatogenesis, with dramatic morphological changes and a final step of spermiation, which involves protein degradation and the removal of excess cytoplasm; therefore, we hypothesized that macroautophagy/autophagy might be involved in the process. To test this hypothesis, we examined the function of ATG5, a core autophagy protein in male germ cell development. Floxed Atg5 and Stra8- iCre mice were crossed to conditionally inactivate Atg5 in male germ cells. In Atg5flox/flox; Stra8- iCre mutant mice, testicular expression of the autophagosome marker LC3A/B-II was significantly reduced, and expression of autophagy receptor SQSTM1/p62 was significantly increased, indicating a decrease in testicular autophagy activity. The fertility of mutant mice was dramatically reduced with about 70% being infertile. Sperm counts and motility were also significantly reduced compared to controls. Histological examination of the mutant testes revealed numerous, large residual bodies in the lumen of stages after their normal resorption within the seminiferous epithelium. The cauda epididymal lumen was filled with sloughed germ cells, large cytoplasmic bodies, and spermatozoa with disorganized heads and tails. Examination of cauda epididymal sperm by electron microscopy revealed misshapen sperm heads, a discontinuous accessory structure in the mid-piece and abnormal acrosome formation and loss of sperm individualization. Immunofluorescence staining of epididymal sperm showed abnormal mitochondria and acrosome distribution in the mutant mice. ATG5 was shown to induce autophagy by mediating multiple signals to maintain normal developmental processes. Our study demonstrated ATG5 is essential for male fertility and is involved in various aspects of spermiogenesis.Abbreviations: AKAP4: a-kinase anchoring protein 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG10: autophagy-related 10; ATG12: autophagy-related 12; cKO: conditional knockout; DDX4: DEAD-box helicase 4; MAP1LC3/LC3/tg8: microtubule-associated protein 1 light chain 3; PBS: phosphate-buffered saline; PIWIL2/MILI: piwi like RNA-mediated gene silencing 2; RT-PCR: reverse transcription-polymerase chain reaction; SQSTM1/p62: sequestosome 1; TBC: tubulobulbar complexes; WT: wild type.

Dataset Information

Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

Publications

Tubulin-binding cofactor E-like (TBCEL), the protein product of the <i>mulet</i> gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

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