Meiotic activation of Mitf/TFEB declines with age in the Drosophila testis

Abstract

Lysosome activity regulates germline development in multiple species. In the Drosophila testis, lysosomes activate as germ cells exit mitosis and enter meiosis. Notably, reduced activity of germ-cell lysosomes, which is seen during aging, leads to fewer viable sperm. Here, we investigated the activity of Mitf/TFEB, a master regulator of lysosome biogenesis, during Drosophila spermatogenesis. We discovered that Mitf activity was upregulated in meiotic-stage spermatocytes, consistent with the lysosome-activation pattern. However, Mitf activity in spermatocytes declined in older males, concurrent with reduced expression of a Mitf-targeted V-ATPase component. These findings provide insight into the regulation of upstream lysosome controls during spermatogenesis.

Figure 1. Developmental and age-related regulation of Mitf activity in the Drosophila testis

A . Schematic of the Drosophila testis. Stem cells reside in the hub (yellow) at the apical tip. Germline stem cells produce a spermatogonium, which undergoes four mitotic divisions. Subsequently, germ cells enter meiosis, producing 16 spermatocytes. Somatic cyst cells (gray lines) encapsulate developing germ cells within individual cysts. Lysosomes (red) activate at meiosis. B . Schematic of the 4Mbox-GFP transgene. When present and active, Mitf binds to repetitive Mboxes, inducing GFP expression. C, D. Representative images of DNA (Hoechst) and 4Mbox-GFP (Mitf activity reporter) in the young-adult Drosophila testis. Enlarged regions given for spermatogonia and spermatocytes in C are outlined in D. A cyst cell nucleus is outlined in C. The mitosis-to-meiosis transition is demarcated by a dashed line in D. E. Quantification of 4MBox-GFP intensities in mitotic spermatogonia ( n =50 spermatogonia from 10 total testes) and meiotic spermatocytes ( n =50 spermatocytes from 10 total testes) in young adult males. Mean ± S.D. p < 0.0001, Mann-Whitney U-test. F. Quantification of 4Mbox-GFP intensities in spermatocytes of young ( n =60 spermatocytes from 12 total testes) and old adult males ( n =80 spermatocytes from 16 total testes). Mean ± S.D. p < 0.0001, Welch's t-test. G. Representative images of DNA (Hoechst) and 4Mbox-GFP (Mitf activity reporter) in spermatocytes of young and old adult males. H. Quantification of VhaSFD-GFP intensities in spermatocytes of young ( n =60 spermatocytes from 12 total testes) and old adult males ( n =65 spermatocytes from 13 total testes). Mean ± S.D. p < 0.0001, Welch's t-test. I. Representative images of DNA (Hoechst) and VhaSFD-GFP in spermatocytes of young and old adult males. Bars, 20 µm.