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. 2018 Sep 27;19(10):2949.
doi: 10.3390/ijms19102949.

Expression of the O-Glycosylation Enzyme GalNAc-T3 in the Equatorial Segment Correlates with the Quality of Spermatozoa

Marie B Nygaard  1   2 , Amy S Herlihy  3 , Charlotte Jeanneau  4 , John E Nielsen  5   6 , Eric Paul Bennett  7 , Niels Jørgensen  8   9 , Henrik Clausen  10 , Ulla Mandel  11 , Ewa Rajpert-De Meyts  12   13 , Kristian Almstrup  14   15
Affiliations

Expression of the O-Glycosylation Enzyme GalNAc-T3 in the Equatorial Segment Correlates with the Quality of Spermatozoa

Marie B Nygaard et al. Int J Mol Sci. .

Abstract

We question whether the expression of GalNAc-T3, the only known O-GalNAc-transferase present in germ cells, is correlated with qualitative and functional parameters of spermatozoa. We investigated the expression of GalNAc-T3 in ejaculated spermatozoa with immunocytochemistry in swim-up purified and acrosome-reacted spermatozoa from quality-control semen donors and in semen samples from 206 randomly selected men representing a broad spectrum of semen quality. Using donor ejaculates and immunofluorescence detection we found that expression of GalNAc-T3 and the presence of the immature O-glycans Tn and T localized to the equatorial segment of spermatozoa. The proportion of GalNAc-T3-positive spermatozoa in the ejaculate increased after swim-up and appeared unaffected by induction of acrosomal exocytosis. The fraction of spermatozoa with equatorial expression of GalNAc-T3 correlated with classical semen parameters (concentration p = 9 × 10-6, morphology p = 7 × 10-8, and motility p = 1.8 × 10-5) and was significantly lower in men with oligoteratoasthenozoospermia (p = 0.0048). In conclusion, GalNAc-T3 was highly expressed by motile spermatozoa and the expression correlated positively with the classical semen parameters. Therefore, GalNAc-T3 expression seems related to the quality of the spermatozoa, and we propose that reduced expression of GalNAc-T3 may lead to impaired O-glycosylation of proteins and thereby abnormal maturation and reduced functionality of the spermatozoa.

Keywords: GALNT3; GalNAc-T3; Male fertility; Mucin-type O-linked glycosylation; Semen quality; Spermatozoa.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results

Figures

Figure 1
Figure 1
Localization of GalNAc-T3 in human spermatozoa. Top panel: Digital Interference Contrast (DIC) images of human spermatozoa. Lower panel: Immunofluorescence (IF) images of GalNAc-T3 (mAb UH5, 2D10) and simple mucin-type O-glycosylation Tn (mAb 5F4) and T (mAb 3C9). Nuclei of the spermatozoa were stained with 4’,6-diamidino-2-phenylindole (DAPI). GalNAc-T3, Tn, and T antigen all localised to the equatorial segment of sperm cells as indicated by the arrowhead. The T antigen was also present in the acrosomal cap (arrow). Scalebar 10 µm.
Figure 2
Figure 2
Expression of GalNAc-T3 on spermatozoa in swim-up fractions and after acrosomal exocytosis. (A) Ejaculates from 4 laboratory quality control (QC)-donors were evaluated for equatorial GalNAc-T3 expression before and after swim-up selection. Three of the samples were further evaluated after incubation with the ionophore ionomycin, which induces the acrosome reaction. (B) Bar plot showing the fold enrichment of GalNAc-T3-positive spermatozoa in the motile and the acrosome-reacted fractions relative to the raw ejaculate. Error bars indicate the standard error of the mean. (C) Representative immunofluorescence images of GalNAc-T3 expression detected with mAb 2D10 in the raw ejaculate, the motile fraction and the acrosome-reacted fraction. Nuclei of the spermatozoa were stained with DAPI. Scale bar 20 µm.
Figure 2
Figure 2
Expression of GalNAc-T3 on spermatozoa in swim-up fractions and after acrosomal exocytosis. (A) Ejaculates from 4 laboratory quality control (QC)-donors were evaluated for equatorial GalNAc-T3 expression before and after swim-up selection. Three of the samples were further evaluated after incubation with the ionophore ionomycin, which induces the acrosome reaction. (B) Bar plot showing the fold enrichment of GalNAc-T3-positive spermatozoa in the motile and the acrosome-reacted fractions relative to the raw ejaculate. Error bars indicate the standard error of the mean. (C) Representative immunofluorescence images of GalNAc-T3 expression detected with mAb 2D10 in the raw ejaculate, the motile fraction and the acrosome-reacted fraction. Nuclei of the spermatozoa were stained with DAPI. Scale bar 20 µm.
Figure 3
Figure 3
Association between the fraction of GalNAc-T3-positive spermatozoa in the ejaculate and classical semen parameters; sperm concentration, morphology and motility. Scatter plots of the fraction of GalNAc-T3-positive spermatozoa and (A) sperm concentration (4th-root transformed), (C) the fraction of spermatozoa with normal morphology (square root transformed), and (E) the fraction of progressive motile spermatozoa (un-transformed). In each scatter plot, the Pearson correlation quotient (r) is indicated together with the p-value of the association. The red line indicates the best linear fit and the grey shaded area the confidence interval. The fraction of GalNAc-T3-positive spermatozoa were divided into quartile-bins (1: Lowest and 4: Highest) and plotted against (B) sperm concentration (4th root-transformed), (D) the fraction of spermatozoa with normal morphology (square root (Sqrt) transformed), and (F) the fraction of progressive motile spermatozoa (un-transformed). p-values of individual comparisons (Wilcoxon rank sum tests) are indicated on each plot together with the overall significance of the grouping (Kruskal-Wallis test).
Figure 4
Figure 4
Association between GalNAc-T3 levels and semen quality. (A) The samples were divided into categories of high (n = 39), intermediate (n = 104), and low (n = 62) semen quality based on parameters given previously [23] and plotted in a boxplot showing the fraction of GalNAc-T3-positive spermatozoa. (B) Boxplot of the fraction of GalNac-T3-positive spermatozoa and the cumulative number of sperm defects as defined by the World Health Organisation (WHO). (C) Boxplot of the fraction of GalNAc-T3-positive spermatozoa and the type of semen deficiency observed. p-values from comparisons of individual groups by Wilcoxon rank sum tests, as well as the overall significance of the grouping by Kruskal-Wallis tests are indicated on each plot.
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