Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: Onset of morphological changes in Mgat1 [F/F]:Stra8-iCre testes. ( A ) Representative control Mgat1 [F/F] and Mgat1 [F/F]:Stra8-iCre testis sections stained with hematoxylin and eosin at 15–28 dpp (n = 3–4 mice per genotype; 20–30 tubules observed per section). At 22 dpp round spermatids were present in control and Mgat1 cKO tubules (arrows). At 25 dpp, few elongated spermatids were seen in Mgat1 cKO tubules and some MNC were observed in a few tubules (asterisks, inset). At 28 dpp, Mgat1 cKO tubules contained MNC comprised of fused spermatids (asterisks, inset and Supplementary Fig. ). Images were scanned at 40× in a Perkin Elmer Scanner. ( B ) Sertoli cells were identified by anti-Sox9 Ab, spermatogonia by anti-PCNA Ab; primary spermatocytes by anti-Sycp3, and acrosomes by PAS. Histograms show numbers of stained cells per 20–30 tubules per section, and numbers of mice examined. Tubules at stage VI and beyond based on morphology with PAS+ acrosomes were counted per 50 tubules. Histograms represent mean ± SEM. Images were photographed at 20×. Controls for antibody specificity are shown in Supplementary Fig. .

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Staining

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: Gene expression changes in Mgat1 cKO germ cells. ( A ) Western blot analysis for basigin before and after endoglycosidase H treatment of germ cell lysates. Basigin from Mgat1 cKO germ cells was sensitive to Endo H digestion, consistent with a lack of complex N-glycans. ( B ) Hierarchical clustering of genes expressed in control versus Mgat1 cKO germ cell cDNA preparations from 22 dpp males, and from 3 replicate cDNA preparations of control versus Mgat1 cKO germ cell RNA pools obtained from 6 mice per genotype at 23 dpp. Red indicates high and green indicates low relative expression. ( C ) qRT-PCR validation of up-regulated genes in microarray data using cDNA from individual preparations used in microarray experiments. Bars represent mean ± SEM (gray, control; black Mgat1 cKO). Data are from two experiments performed in duplicate (n = 3 mice/group). * p < 0.05, ** p < 0.01. ( D ) qRT-PCR of down-regulated genes identified in microarray data performed on the same RNA samples used in B. Data are from two experiments performed in duplicate (n = 3 mice/group). * p < 0.05, ** p < 0.01. Both control and Mgat1 cKO transcripts were determined relative to Actb .

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Microarray

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: IPA of DEGs in control versus Mgat1 cKO germ cells. ( A ) Canonical pathways significantly overrepresented in Mgat1 cKO germ cells compared to control according to their -log p value. Colors indicate the activation Z score of processes: activated processes are orange, while inhibited processes are blue. No activity pattern is represented by gray, and no change is denoted by white. Right panel shows comparison analysis of canonical pathways from independent 22 dpp versus 23 dpp mutant germ cells based on p value. ( B ) Comparison of biological functions based on p value in 22 versus 23 dpp shows Cellular Function and Maintenance and Reproductive System Development and Function as the top hits in both 22 and 23 dpp DEGs. ( C ) IPA upstream regulators of DEGs in control versus Mgat1 cKO 22 dpp germ cells. Symbols of target proteins or upstream regulators in red indicate a predicted increase or activation. The symbol shapes denote the molecular classes of proteins. Right panel shows the heat map for comparison of upstream regulators at 22 and 23 dpp based on activation Z score. ( D ) IPA downstream effect analysis networks associated with diseases and functions. Z scores for both diseases are negative indicating a reduction in disease. Edges and nodes are color-coded based on the predicted relationship as indicated in the legend. Right panel shows the heat map for comparison of disease conditions at 22 and 23 dpp based on activation Z score.

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Activation Assay, Activity Assay, Mutagenesis, Cell Function Assay

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: The top network identified by IPA in 22 dpp Mgat1 cKO germ cells. ( A ) Molecules are represented as nodes (see legend). Nodes in red or green represent up-regulated or down-regulated genes, respectively. ( B ) Validation of Network 1 by qRT-PCR performed in duplicate on germ cell cDNA prepared from 3 mice in each group. Gray bars represent control and black bars represent Mgat1 cKO germ cells (mean ± SEM; * p < 0.05, ** p < 0.01).

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Quantitative RT-PCR

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of microarray data in 22 dpp Mgat1 cKO germ cells. GSEA enrichment plots of three gene clusters that are enriched in Mgat1 cKO germ cells were ( A ) Gamete Generation, ( B ) Matzuk Spermatid Differentiation, and ( C ) Matzuk Spermatozoa Formation. Heat map of positively-enriched genes in Mgat1 cKO germ cells compared to control for ( D ) gamete generation ( E ) Matzuk spermatid differentiation and ( F ) Matzuk spermatozoa formation. *Genes involved in spermiogenesis that are up-regulated prematurely in Mgat1 cKO males at 22 dpp.

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Microarray

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: Signaling pathways in Mgat1 cKO germ cells at 22 dpp. ( A ) GSEA analysis showing enrichment of a PDGF_ERK signature in control germ cells. ( B ) Heat map shows the cluster of DEGs in the PDGF_ERK signaling pathway positively-enriched in control versus Mgat1 cKO germ cells. Arrow identifies PDGFRA as down-regulated in Mgat1 cKO germ cells. (C) Western blot analysis of phosphorylated and unphosphorylated ERK1, ERK2 and AKT in germ cells of Mgat1 cKO compared to control. The gels from which these data were obtained are shown in Supplementary Fig. . ( D ) Histogram of 2–3 independent experiments analyzed in 4–6 gels is shown (mean ± SEM; *** p < 0.005, **** p < 0.001).

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: CX43 in 28 dpp control and Mgat1 cKO testes. ( A ) IHC of CX43 expressed in 28 dpp control and Mgat1 cKO testis sections were scored blindly as score 1, score 2, or score 3 to reflect poor, medium or high staining, respectively. ( B ) Scores from 90 CX43-positive tubules were counted per genotype. Mean ± SEM, ** p < 0.01.

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Staining

Journal: Scientific Reports

Article Title: MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis

doi: 10.1038/s41598-018-20465-3

Figure Lengend Snippet: Basigin signaling is reduced in Lec1 CHO cells, a model for Mgat1 cKO germ cells. Flow cytometry profiles of CHO wild type ( A ) and Lec1 mutant ( B ) CHO cells and basigin transfectants. Also shown is a complex N-glycan typical of CHO cells and the oligomannosyl N-glycan substrate of MGAT1 that accumulates on glycoproteins of Lec1 CHO cells. Blue square, GlcNAc; green circle, Man; yellow circle, Gal; red triangle, fucose; purple diamond, sialic acid. ( C ) CHO wild type, Lec1 and basigin transfectant lysates following 24 hr serum starvation and a 15 min incubation in serum-free medium were analyzed by western blotting using ERK Abs as shown. The gels from which these data were obtained are shown in Supplementary Fig. . ( D ) The pERK1/ERK1 and pERK2/ERK2 values for CHO, Lec1 and respective basigin transfectants (left panel), and the ratio of these values in basigin expressing versus non-basigin expressing CHO and Lec1 cells (right panel). Data are mean ± SEM from 9–13 gels of lysates run in 6 independent experiments. * p < 0.05 in ( D ) left panel is based on a one-tailed, unpaired Student’s t test with Welch’s correction; in ( E , left panel) significance is based on the non-parametric, two-tailed Wilcoxon matched-pairs signed rank test; in ( E ) right panel it is based on the unpaired, two-tailed Student’s t test with Welch’s correction. ** p < 0.01, *** p < 0.005 and **** p < 0.001.

Article Snippet: CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C) were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent.

Techniques: Flow Cytometry, Mutagenesis, Transfection, Incubation, Western Blot, Expressing, One-tailed Test, Two Tailed Test