Expression, Localization of SUMO-1, and Analyses of Potential SUMOylated Proteins in Bubalus bubalis Spermatozoa

Abstract

Mature spermatozoa have highly condensed DNA that is essentially silent both transcriptionally and translationally. Therefore, post translational modifications are very important for regulating sperm motility, morphology, and for male fertility in general. Protein sumoylation was recently demonstrated in human and rodent spermatozoa, with potential consequences for sperm motility and DNA integrity. We examined the expression and localization of small ubiquitin-related modifier-1 (SUMO-1) in the sperm of water buffalo (Bubalus bubalis) using immunofluorescence analysis. We confirmed the expression of SUMO-1 in the acrosome. We further found that SUMO-1 was lost if the acrosome reaction was induced by calcium ionophore A23187. Proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were identified, including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets. The expression of SUMO-1 in the acrosome region of water buffalo sperm and the identification of potentially SUMOylated proteins important for sperm function implicates sumoylation as a crucial PTM related to sperm function.

Keywords: Bubalus bubalis; SUMO-1; post translational modification; protein; spermatozoa.

Figure 1

Acrosome identification in buffalo bull sperm and four categories of Buffalo bull sperm acrosome status. (A) (Image a) Coomassie Brilliant Blue staining of sperm with an intact and reacted acrosome, (image b) FITC-PNA stained, (image c) DAPI-stained, (image d) DAPI+FITC-PNA- stained images. (B) (Image a) intact acrosome, (image b) equatorial acrosome region, (image c) post acrosomal region, (image d) reacted acrosome. Scale bar, 20 μm; Magnification, 1,000x.

Figure 2

Localization of SUMO-1 in the acrosome of Buffalo bull and mouse spermatozoa. (A) DAPI, for nuclear staining (blue signal), SUMO-1, Anti-SUMO1 antibody followed by a CY3-conjugated secondary antibody (Red fluorescence), Merged, DAPI nuclear staining plus anti-SUMO1 antibody. (B) SUMO1 co-localization and expression in buffalo bull sperm. Collected spermatozoa were stained with SUMO1 antibody pursued by incubation with Cy-3 conjugated goat anti rabbit IgG (color red). The specimen were then restained with 4, 6-diamino-2-phenylindole (DAPI, 1:5,000 in PBS) (blue color) and FITC-PNA (flurescein isothiocyanate-conjugated peanut agglutinin) (green color). In (panel a) the superposed images shown in the spermatozoa were stained with SUMO-1 antibody followed by incubation with Cy-3 conjugated goat anti rabbit IgG. (panel b) the superposed images are shown in negative samples as control. (panel c) the images show both an intact and reacted acrosome, and the disappearance of SUMO-1 in the acrosome-reacted buffalo bull spermatozoa. Note that both SUMO-1 and FITC-PNA disappeared in acrosome region of the acrosome-reacted spermatozoa. The images were observed using a Nikon inverted fluorescence microscope. The scale bar is 100 μm.

Figure 3

Expression of SUMO-1 in water buffalo spermatozoa. (A) Western blot analysis of SUMO-1 expression in water buffalo spermatozoa after in vitro capacitation, showing capacitation-induced changes in SUMO-1-modifications in sperm (lane b) compared to non-capacitated sperm (lane a). (B) Western blot analysis of acrosome reacted (lane a) vs. acrosome non-reacted (lane b) water buffalo spermatozoa. (C) Buffalo bull sperm proteins were immunoprecipatated with SUMO-1 antibody, and then run for SDS-PAGE and stained with coomassive brilliant blue. For the negative control (NC). (D) SUMO-1 conjugates were pull down from water buffalo sperm lysates by a SUMO-1 antibody and were analyzed by western blot to detect SUMO-1 expression.

Figure 4

SCL in whole cell sperm in Bubalus bubalis. Plot describing the distribution of protein Sub-cellular localization [SCL] predicted by iLoc-Animal and iLoc-Euk tools. [C]: cytoplasm; [C,N]: cytoplasm and nculeus; [C,EC]: cytoplasm and extracellular; [CM,C]: cell membrane and cytoplasm; [C,MT]: cytoplasm and mitochondrion; [C,ED]: cytoplasm and endosome; [C,ER]: cytoplasm and endoplasmic reticulum; [C,N,CK]: cytoplasm, nucleus, cytoskeleton; [C,N,ER,MT]: cell membrane, cytoplasm, endoplasmic reticulum, and mitochondrion; [NA]: No Available results for those sequences.

Figure 5

Functional Protein Classification (COGs). (A) Distribution of proteins count in the COGs categories. Colored boxes correspond to the unique COGs subcategories (EggNOG v4.5 terms). (B) The pie shown each COG category percentage.

Figure 6

GO analysis. The GO analysis was performed with PANTHER (V11). (A) Details of GO terms distribution. (B) The terms were reclassified using Categorizer [http://animalgenome.org]. This represents the mapping of 22 GO terms to 127 of the “GO Slim” ancestor terms by single count. (C) Plot of GO terms details. Further analysis of GO terms was conducted by Rivgo [http://revigo.irb.hr].

Figure 7

Protein-Protein Interaction [STRING-V10.0] (PPIs). The PPI was built using the following parameters: Bos taurus background (No match for Bubalus bubalis found, B.bubalis genome is not full and well-annotated yet). Highest confidence Score = 0.55. The dark line in the graph correspond to Highest confidence score higher than 0.7. Hide disconnected nodes in the network. PPI enrichment p-value = 0.000124, clustering coefficient: 0.902.

Figure 8

Distribution of SUMO modification types reported by (GPS-SUMO2.0).

Figure 9

Seq2Logo2.0 representation of three types of SUMO modification peptides sequences reported by (GPS-SUMO2.0).

Figure 10

TPR and Filamin-C analysis with [STRING-V10.0]. Protein of interest is marked in Red in the graph. (A) TPR protein-protein interaction network. (Highest confidence Score = 0.9. Hide disconnected nodes in the network. PPI enrichment p-value = 0); (B) Filamin-C, COG analysis. This protein is marked as Non Supervised Orthologous Group (NOG19963) and is below to three edges of the graph with two COGs terms [COG5160 andCOG5079]. (C) COG analysis for TPR protein with string. TPR is marked as Non Supervised Orthologous Group (NOG07190). Two COGs terms are present in the graph [COG5646 andCOG5201].