Enrichment of lactate dehydrogenase C (LDHC) and phosphoglycerate kinase (PGK2) in cytoplasmic droplets (CDs) and CD-dependent localization of LDHC and PGK2 in epididymal spermatozoa.

<p>(<b>A</b>) A representative SDS-PAGE gel showing equal loading of proteins (8 μg/lane) isolated from total epididymal spermatozoa (T), epididymal spermatozoa after CD removal (R), purified CDs (CD) and heart (H). M, protein size marker. (<b>B</b>) A representative Western blot result showing levels of LDHC in total epididymal spermatozoa (T), epididymal spermatozoa after CD removal (R), purified CDs (CD) and heart (H). (<b>C</b>) A representative Western blot result showing levels of PGK2 in total epididymal spermatozoa (T), epididymal spermatozoa after CD removal (R), purified CDs (CD) and heart (H). (<b>D</b>) Immunofluorescent staining of LDHC and PGK2 in CD-bearing (upper panels), CD-free (middle panels) epididymal spermatozoa form the caput, and epididymal spermatozoa after centrifugation-based CD removal (lower panels). All panels were in the same magnification (Scale bar, 5μm). </p

CDs appear to be required for normal mitochondrial activation and ATP production during epididymal sperm maturation.

<p>(<b>A</b>) Mitochondrial membrane potential in murine caput, corpus and cauda epididymal spermatozoa with or without CDs. Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=5). (<b>B</b>) ATP levels in murine caput, corpus and cauda epididymal spermatozoa with or without CDs. Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=5). (<b>C</b>) Mitochondrial membrane potential in monkey caput, corpus and cauda epididymal spermatozoa with or without cytoplasmic droplets (CDs). Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=5). (<b>D</b>) ATP levels in monkey caput, corpus and cauda epididymal spermatozoa with or without CDs. Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=5). (<b>E</b>) Pyruvate levels in murine caput, corpus and cauda epididymal spermatozoa with or without CDs. Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=5). (<b>F</b>) Effects of 5mM Ammonium Oxalate (AO) and 1mM α-Chlorohydrin (ACH) on the development of progressive motility of murine caput, corpus and cauda epididymal spermatozoa. Data are presented as mean ± SEM. Bars with different letters are significantly different (p<0.05, n=4).</p

RAN-Binding Protein 9 is Involved in Alternative Splicing and is Critical for Male Germ Cell Development and Male Fertility

<div><p>As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages <i>in vitro</i>, and this is further supported by the neonatal lethality phenotype in <i>Ranbp9</i> global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of <i>Ranbp9</i> specifically in testicular somatic and spermatogenic cells, we discovered that <i>Ranbp9</i> was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of <i>Ranbp9</i>. Our data uncovered a novel role of <i>Ranbp9</i> in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.</p></div

<i>Ranbp9</i> deficiency causes male germ cell apoptosis and DNA double-strand breaks.

<p>(A) TUNEL assays on WT, <i>Ranbp9</i> global KO (<i>Ranbp9^Δ/Δ</i>) and gcKO testes. Arrows point to apoptotic cells stained in brown. Scale bar = 50 µm. Significantly increased average number of apoptotic cells is observed in both <i>Ranbp9</i>^Δ/Δ and gcKO testis (the far right panel). >60 cross-sections were scored for the average number of apoptotic cells per tubule for each genotype. Three mice of each genotype were analyzed, and data were presented as mean ± SD, n = 3. (B) Immunofluorescence staining of γH2AX in seminiferous tubules of WT and gcKO testes at ∼stage VI. In WT seminiferous tubules, γH2AX immunoreactivity is mostly confined to the XY body (arrows) in pachytene spermatocytes and completely absent in round spermatids (arrowheads). In contrast, in gcKO seminiferous tubules, numerous round spermatids exhibit strong γH2AX staining (arrowheads) in addition to its normal localization in the XY body (arrow) in pachytene spermatocytes. (C) qPCR analyses showing significantly reduced levels of <i>Prm1</i>, <i>Prm2</i>, <i>Tnp1</i> and <i>Tnp2</i> mRNAs in 6-week old <i>Ranbp9</i> gcKO testes. Data are presented as mean ± SEM, n = 3.</p

Expression profiles of <i>Ranbp9</i> during testicular development and spermatogenesis in mice.

<p>(A) qPCR analyses of <i>Ranbp9</i> mRNA levels in multiple organs in mice. Data are presented as mean ± SEM, n = 3. (B) Expression of <i>Ranbp9</i> and <i>Ranbp10</i> during postnatal testicular development. Levels of <i>Ranbp9</i> and <i>Ranbp10</i> mRNAs in developing testes at postnatal day 7 (P7), P14, P21, P28, P35, and in adult (Ad) were analyzed using qPCR. Data are presented as mean ± SEM, n = 3. (C) Expression of RANBP9 protein during postnatal testicular development. Levels of RANBP9 in the testes from newborn (P0), postnatal day 3 (P3), P7, P14, P21, P28, and P35 and adult male mice were determined using western blot analyses. ACTIN was used as a loading control. (D) Immunofluorescent detection of RANBP9 in homozygous <i>Ranbp9</i> flox (<i>Ranbp9^lox/lox</i>) and <i>Ranbp9</i> global knockout (<i>Ranbp9^Δ/Δ</i>) testes. In <i>Ranbp9^lox/lox</i> testes, RANBP9 immunoreactivity was mostly detected in the nucleus of spermatocytes (spc) and spermatids (spd). Insets show the digitally magnified view of the framed area. RANBP9 was also detected in the nucleolus of Sertoli cells (Ser), and in both the cytoplasm and the nucleus in interstitial Leydig cells (Ley) (Middle panels). While the nucleus was partially RANBP9-positive in a subpopulation of spermatogonia (spg), RANBP9 staining covered the entire nucleus in both pachytene spermatocytes (pachy) and round spermatids (rspd) (Lower panels). In <i>Ranbp9^Δ/Δ</i> testes, RANBP9 staining was completely absent. Scale bar = 50 µm.</p

Schematic diagram showing the proposed model of RANBP9 function during spermatogenesis in mice.

<p>In the nuclei of spermatocytes (including leptotene, zygotene, pachytene and diplotene) and spermatids, RANBP9 binds key splicing factors (e.g. SF3B3, HNRNPM), and poly(A) binding proteins (PABPC1/2), to coordinate proper alternative splicing of its target mRNA transcripts. Correctly spliced, mature mRNAs are subsequently exported to the cytoplasm to function in spermatocytes and spermatids. Green arrows denote export of processed mRNAs from the nucleus to the cytoplasm.</p

Disruptions of the mRNA transcriptome and alternative splicing patterns in <i>Ranbp9</i> gcKO testes.

<p>(A) Scatter plot showing significantly de-regulated transcripts in <i>Ranbp9</i> gcKO testes compared to WT controls. Blue dots (2,313) represent significantly upregulated transcripts, while red dots (316) denote significantly downregulated transcripts (p<0.05, fold change>2). Yellow dots illustrate unchanged transcripts. (B) Venn diagram showing the number of unique transcript isoforms detected in <i>Ranbp9</i> gcKO (2,420) and WT (277) testes. (C) Distribution of 1,816 aberrant splicing events (insertions or deletions) along the entire length of mRNAs in gcKO testes. The y-axis represents the size of insertions (positive values) or deletions (negative values), whereas the x-axis denotes location percentage (splicing location/total transcript size), reflecting the relative position of splicing events along the entire length of the transcripts, e.g., 0% refers to the very 3′end, 50% means the middle of the transcript and 100% indicates the very 5′end. (D) Semi-qPCR-based detection of aberrant alterative splicing patterns in three RANBP9 direct target mRNAs (<i>Rfx2</i>, <i>Plec</i> and <i>Usp19</i>). Lower panels represent the schematic diagram of alternatively spliced exons detected by RNA-Seq analysis. <i>Gapdh</i> was used as a loading control.</p

RANBP9 binds numerous mRNAs and affects their expression levels at least partially through affecting alternative splicing.

<p>(A) A representative mRNA assembly output showing RIP-Seq reads for <i>Ddx25</i> identified from the RIP products using the RANBP9 antibody and IgG (control). (B) qPCR analyses of levels of three RANBP9-bound mRNAs (<i>Rfx2</i>, <i>Plec</i> and <i>Usp19</i>) in WT and gcKO testes. All three are highly enriched in WT compared to gcKO testes, demonstrating the specificity of the anti-RANBP9 antibody used in RIP-Seq assays. (C) GO enrichment analyses of RANBP9-bound mRNAs identified using RIP-Seq. (D) Venn diagram showing the number of up- and down-regulated RANBP9-bound target transcripts in gcKO testes (P<0.05, fold change>1.5). (E) qPCR analyses of levels of 17 RANBP9 target transcripts in 6-week-old WT and gcKO testes. Data are presented as mean ± SEM, and significantly altered levels were marked with * (n = 4, P<0.05).</p

Identification of RANBP9-interacting partners in murine testes using immunoprecipitation followed by mass spectrometry (IP-MS).

<p>(A) A representative gel image showing bands representing proteins immunoprecipitated by the monoclonal anti-RANBP9 antibody used or IgG (control). Arrows indicate protein bands unique to the IP products of anti-RANBP9 antibody, which were excised for subsequent MS analyses. (B) A list of 18 RANBP9-interacting partners in murine testes identified by IP-MS. All proteins were detected multiple times in all three biological replicates. (C) Results of gene ontology (GO) term enrichment analyses of RANBP9-interacting proteins. (D) Validation of interactions between RANBP9 and four putative RANBP9-interacting proteins (PABPC1, PABPC2, SF3B3 and HNRNPM) in murine testes by <i>in vivo</i> co-immunoprecipitation assays, in which antibodies specific for the four proteins were used for immunoprecipitation (IP) followed by Western blot analyses using a mouse monoclonal anti-RANBP9 antibody. IgG was used as a negative control. (E) A representative Western blot analyses showing levels of four RANBP9-interacting proteins (SF3B3, HNRNPM, PABPC1 and PABPC2) in 6-week old WT and <i>Ranbp9</i> gcKO testes.</p

Additional file 1 of Association of plasma angiogenin with risk of major cardiovascular events in type 2 diabetes

Supplementary Material 1: Supplementary Figures and Table