Contributed equally
Germ cell maturation is essential for spermatogenesis and testis homeostasis. ATP synthase serves significant roles in energy storage in germ cell survival and is catalyzed by alterations in the mitochondrial membrane proton concentration. The intrinsic cellular mechanisms governing stem cell maturation remain largely unknown. In the present study,
The process of spermatogenesis is conserved between
Adenosine 5′-triphosphate (ATP) synthase is an enzyme complex that creates the energy storage molecule ATP, which is commonly used to provide energy for the majority of tissues and organisms (
In
The Upstream Activation Sequence/Gal4 transcription factor (UAS/Gal4) system-based RNA interference (RNAi) silencing method has previously been used to analyze the male biological reproductive process in
All flies were fed with standard corn meal food at 25°C. Information about the alleles and transgenes used in the present study are available either in FlyBase (
Fly crosses and male fertility tests were performed as described in our previous study (
Fly testes were dissected in 1X PBS and fixed at room temperature for 30 min in 4% paraformaldehyde. Following washing three times in 1X PBS with 0.1% Triton X-100 (PBST) and blocking at room temperature for 1 h in 5% bovine serum albumin (Sangon Biotech, Shanghai, China) dissolved in 1XPBS, the samples were incubated with primary antibodies overnight at 4°C. Following washing three times for 10 min in 0.1% PBST, the samples were then incubated for 1 h with secondary antibodies (Alexa Fluor® 488 or Cy™ 3) at room temperature followed by three washes in 0.1% PBST. Testes were then stained with Hoechst 33342 (1.0 mg/ml; Invitrogen; Thermo Fisher Scientific, Inc, Waltham, MA, USA) at room temperature for 5 min prior to mounting.
The antibodies used were as follows: Mouse anti-EYA transcriptional coactivator and phosphatase 1 [Eya; 1:20; eya10H6; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; rat anti-Drosophila E-cadherin homolog (DE-cadherin; 1:20; DCAD2; DSHB); rabbit anti-DEAD-Box helicase 4 (Vasa; 1:1000; cat. no. sc-30210; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Secondary antibodies conjugated to Alexa Fluor® 488-rabbit (cat. no. 711-545-152; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or Cy™ 3-mouse (cat. no. 715-165-150; Jackson ImmunoResearch Laboratories, Inc.) were diluted at 1:1,000.
Diagrams indicating the oxidative phosphorylation process and distribution of the subunits of ATP synthase were adopted from the Kyoto Encyclopedia of Genes and Genomes Database (
Experiments were repeated at least three times. The fertility rate was evaluated for statistical differences using one-way analysis of variance and Least Significance Difference post-hoc test by SPSS software (v22; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
To examine the function of the ATP synthase β subunit in
To additionally explore the role of the ATP synthase β subunit, the structure of the testis and changes in the expression pattern were detected at the molecular level by immunofluorescence. The Eya gene is required for the development of the
Knockdown of the ATP synthase β subunit in early germ cells resulted in a significant decrease in Vasa-positive germ cells in spermatocytes in nos>ATPsyn β RNAi testes [59.10% of testes with defects in ATPsyn β RNAi-1 (n=22); 61.90% of testes with defects in ATPsyn β RNAi-2 (n=21)] compared with WT testes (n=32), but there was no significant difference in the number of GSCs and spermatogonia between the knockdown and WT testes (
To additionally evaluate the biological function of the ATP synthase β subunit, ATPsyn β was knocked down using bam-Gal4. The Bam protein is a key differentiation factor in early germ cells, and it determines the differentiation fate of spermatogonia and triggers spermatocyte development (
In the present study, the function of ATP synthase in
To detect whether other ATP synthase subunits also served significant roles in male fertility and germ cell mature in testes, an
For the male fertility test, it was identified that the fertility levels of the ATPsyn f6 RNAi and ATPsyn α RNAi strains, driven by nos-Gal4, were partially affected (
When nos-Gal4 was used for screening, all the lines exhibited germ cell maturation defects. These testes were stained with the germ cell marker Vasa. In the majority of the ATPsyn RNAi testes, early germ cells, including GSCs and TA-spermatogonia, were Vasa-positive. In addition, certain spermatocytes in a number of ATPsyn RNAi testes were Vasa-negative (8/15 in ATPsyn b RNAi; 9/17 in ATPsyn c RNAi; 6/19 in ATPsyn d RNAi, 10/25 in ATPsyn OSCP RNAi; 11/19 in ATPsyn f6 RNAi; 15/25 in ATPsyn α RNAi; and 13/27 in ATPsyn γ RNAi). ATPsyn RNAi testes were also stained with the somatic cyst cell marker Eya, and all cells were identified to be positive for Eya (
Next, the ability of germ cell maturation in ATP synthase-deficient testes, driven by bam-Gal4, was examined. Similar germ cell maturation defects were identified in bam>ATPsyn RNAi testes: In 48.00% (12/25) ATPsyn b RNAi, 29.17% (7/24) ATPsyn c RNAi, 29.63% (8/27) ATPsyn d RNAi, 44.00% (11/25) ATPsyn OSCP RNAi, 42.11% (8/19) ATPsyn f6 RNAi, 31.82% (7/22) ATPsyn α RNAi and 47.80% (11/23) ATPsyn γ RNAi testes, some of the spermatocytes were Vasa-negative, while the early germ cells and somatic hub and cyst cells were not affected (
The roles of most of the major ATP synthase subunits remain to be elucidated. Analysis of the major ATP synthase subunits driven by nos-Gal4 and bam-Gal4 indicated that ATP synthase subunits may assemble into a complex and participate in germ cell development via oxidative phosphorylation (
The
A previous study indicated that the ATP synthase served to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex in
A previous study has demonstrated that the knotted onions (knon) gene encodes a testis-specific paralog of ATP synthase subunit d, and is required for the internal structure of the nebenkern and its subsequent disassembly and elongation. knon knockout mutants exhibited aberrant mitochondrial elongation during spermatogenesis and faulty nebenkern morphology (
The present study described evidence associating germ cell mature and cell survival to ATP synthase. The data demonstrated the role of a cell biological process in germ cell development, and these results contribute to the knowledge of the role of ATP synthase in ATP production.
In a number of cell types, dimerization of ATP synthase complexes with their axes at a certain angle is important for determining the sharp positive curvature of the inner mitochondrial membrane (
Future studies on ATP synthase subunits and other members of the oxidative phosphorylation system may explore their regulatory network and mechanism underlying the clustered regularly interspersed short palindromic repeats (CRISPR) associated protein 9/CRISPR system in
The authors would like to thank Professor Dahua Chen from Institute of Zoology, Chinese Academy of Sciences for sharing reagents and stocks.
The present study was supported by National Natural Science Foundation of China (grant nos. 31701298 and 81402100), Natural Science Foundation of Jiangsu Province (grant no. BK20170562), Key Research Foundation of Zhenjiang Social Development (grant nos. SH2017013, SH2017020, SH2016028, SH2016031 and SH2014026), Key Research Foundation of Zhenjiang Health Science and Technology (grant no. SHW2016001), Open Fund of State Key Laboratory of Reproductive Medicine of Nanjing Medical University (grant no. SKLRM-KA201603), the Foundation of Health and Family Planning Commission of Jiangsu Province (grant no. Q201408), the Foundation for Young Medical Talents of Jiangsu province (grant no. QNRC2016840), Six Talent Peaks Project in Jiangsu Province (grant no. WSW-007), Science Foundation of Doctorate Research of Affiliated Hospital of Jiangsu University (grant no. jdfyRC2016005), Suzhou Key Medical Center (grant no. SZZX201505), Suzhou Introduced Project of Clinical Medical Expert Team (grant no. SZYJTD201708) and Jiangsu Provincial Medical Innovation Team (grant no. CXTDB2017013).
All data generated or analyzed during this study are included in this published article.
JY, BC and JF conceived and designed the experiments. BZ, CQ, XC, YY and XL performed the experiments. BX, ZH and JL analyzed the data. CS, XH and QS contributed to the fly feeding and part of the data analysis. ML and HL contributed to the fly crosses and male fertility test. HL, JF and JY wrote the manuscript. All authors read and approved the final manuscript.
The present study was approved by Ethics Committee for Biomedical Research at Affiliated Hospital of Jiangsu University.
Not applicable.
The authors declare that they have no competing interests.
Structure of the WT testis in
Fertility rate of flies with ATP synthase β subunit knockdown. A total of two independent RNAi lines of ATPsyn β (ATPsyn β RNAi-1 and ATPsyn β RNAi-2) were used. (A) The fertility rate of WT and nos>ATPsyn β RNAi males. (B) The fertility rate of WT and bam>ATPsyn β RNAi males. Data were evaluated by one-way analysis of variance and Least Significant Difference post-hoc test. *P<0.05, **P<0.01 and ***P<0.001. WT, wild type; ATP, adenosine 5′-triphosphate; RNAi, RNA interference; ATPsyn, ATP synthase; Nos, Nanos; Bam, bag of marbles.
Knockdown of ATP synthase β subunit with nos-Gal4 and bam-Gal4. (A-C) Immunofluorescence of (A) WT, (B) nos>ATPsyn β RNAi-1 and (C) nos>ATPsyn β RNAi-2 testes. (D-F) Immunofluorescence of (D) WT, (E) bam>ATPsyn β RNAi-1 and (F) bam>ATPsyn β RNAi-2 testes. Representative images of WT testes illustrate the tip of the testis with hub cells, GSCs, CySCs, differentiated germ cells and mature cyst cells. Germ, cyst, and undifferentiated germ cells were stained to detect Vasa (green stain), Eya (red stain), and DNA (blue stain). Areas enclosed by white lines represent regions of germ cells with maintenance defects. Scale bars, 20 µm WT, wild type; ATP, adenosine 5′-triphosphate; ATPsyn, ATP synthase; Nos, Nanos; Bam, bag of marbles; RNAi, RNA interference; *, hub cells; Vasa, DEAD-Box helicase 4; Eya, EYA transcriptional coactivator and phosphatase 1.
Fertility rate of knockdown of major ATP synthase subunits. (A) The fertility rate of WT and major ATP synthase subunit RNAi knockout flies driven by nos-Gal4. (B) The fertility rate of WT and major ATP synthase subunit RNAi knockouts driven by bam-Gal4. Data were evaluated by one-way analysis of variance and Least Significant Difference post-hoc test. *P<0.05, **P<0.01 and ***P<0.001. ATP, adenosine 5′-triphosphate; ATPsyn, ATP synthase; Nos, Nanos; Bam, bag of marbles; WT, wild type; RNAi, RNA interference; OSCP, oligomycin sensitivity-conferring protein; n.s., no statistical difference.
Knockdown of major ATP synthase subunits in germ cells. (A-G) Immunofluorescence of ATPsyn RNAi testes driven by nos-Gal4, including (A) b, (B) c, (C) d, (D) f6, (E) α, (F) γ and (G) OSCP. (H-N) Immunofluorescence of ATPsyn subunits in RNAi testes driven by bam-Gal4, including (H) b, (I) c, (J) d, (K) f6, (L) α, (M) γ and (N) OSCP. Areas enclosed by white lines represent regions of germ cells with maintenance defects. Scale bars, 20 µm. *, hub cells; ATP, adenosine 5′-triphosphate; ATPsyn, ATP synthase; Nos, Nanos; Bam, bag of marbles; WT, wild type; RNAi, RNA interference; OSCP, oligomycin sensitivity-conferring protein; Vasa, DEAD-Box helicase 4; Eya, EYA transcriptional coactivator and phosphatase 1.
Assembly of ATP synthase and GO analysis of ATP synthase subunits. (A) A Kyoto Encyclopedia of Genes and Genomes pathway map indicates that ATP synthase assembles into a complex and is involved in the development of germ cells by oxidative phosphorylation. Electron transport through complexes I–IV drives the extrusion of protons into the intermembrane space. The proton gradient generated is harnessed by the ATP synthase to drive the synthesis of ATP. (B) The colored boxes indicate which ATP synthase components were screened. (C) Clusters identified by the Database For Annotation, Visualization, and Integrated Discovery bioinformatics database indicate selected GO terms of genes identified in the ATP synthase screening process. ATP, adenosine 5′-triphosphate; GO, Gene Ontology.
ATPsyn RNA interference strains used in the screening process.
ATPsyn | THFC no. | BDSC no. | TRiP no. | Annotation symbol | Gene symbol | Hairpin ID |
---|---|---|---|---|---|---|
b | THU2903 | 28062 | JF02899 | CG8189 | ATPsynB | TR02373P.1 |
c | THU0360 | 35464 | GL00390 | CG1746 | ATPsynC | SH01517.N2 |
d | THU1424 | 33740 | HMS01078 | CG6030 | ATPsynD | SH01691.N |
OSCP | TH01379.N2 | – | – | CG4307 | ATPsynO | SH03719.N2 |
f6 | TH01666.N | – | – | CG4412 | ATPsynCF6 | SH04247.N |
α | THU2900 | 28059 | JF02896 | CG3612 | ATPsyn-α | TR02362P.1 |
β-1 | THU2798 | 27712 | JF02792 | CG11154 | ATPsyn-beta | TR02374P.1 |
β-2 | THU2896 | 28056 | JF02892 | CG11154 | ATPsyn-beta | TR02347P.1 |
γ | THU3092 | 28723 | JF03150 | CG7610 | ATPsyn-gamma | TR02371P.1 |
ATP, adenosine 5′-triphosphate; THFC, TsingHua Fly Center; BDSC, Bloomington Drosophila Stock Center; OSCP, oligomycin sensitivity-conferring protein; TRiP, The Transgenic RNAi Project.