Deletion of Mgat2 in spermatogonia blocks spermatogenesis

Abstract

Identifying factors required for spermatogenesis is important for understanding mechanisms of male fertility. Inactivation of either the Mgat1 or Man2a2 gene leads to a block in spermatogenesis causing infertility in male mice. MGAT1 GlcNAc-transferase initiates complex N-glycan synthesis and MAN2A2 mannosidase generates the substrate for MGAT2 GlcNAc-transferase to form a biantennary complex N-glycan. In this paper, we show that conditional deletion of Mgat2 in spermatogonia via Stra8-iCre caused a novel block in spermatogenesis, largely prior to the formation of round spermatids. Mgat2[-/-] germ cells did not bind the lectins Phaseolus vulgaris leucoagglutinin (L-PHA) or Griffonia simplicifolia II (GSA-II), similar to germ cells lacking MGAT1 and complex N-glycans. However, overall spermatogenic defects were distinct in germ cells with deleted Mgat2 versus Mgat1. In addition, RNA-seq analysis at 15 days after birth revealed a unique transcriptomic landscape in Mgat2[-/-] germ cells with genes required for sperm formation and functions being most downregulated. Bioinformatic analyses using the ingenuity pathway analysis (IPA) algorithm identified ERK and AKT as central activities. Western blot analyses of 15-day germ cell lysates confirmed that both AKT and ERK1/2 signaling were increased by loss of MGAT2 in germ cells. By contrast, Mgat1[-/-] germ cells were previously shown to have reduced ERK signaling and unchanged AKT activity. Therefore, since the loss of all complex N-glycans is common to each mutant model, the different immature N-glycans that accumulate in Mgat2[-/-] versus Mgat1[-/-] germ cells are proposed to be the basis of their unique spermatogenic phenotypes.

Keywords: AKT and ERK signaling; MGAT2; complex N-glycans; conditional knockout; spermatogenesis.

FIGURE 1

Deletion of Mgat2 in spermatogonia. (A) Diagram showing the different germ cells in a testis tubule from a typical 7-week C57BL6/J male. Sg, spermatogonium; Sc, spermatocyte; RSt, round spermatid; ESt, elongated spermatid; Sp, sperm; Stra8-iCre is first expressed in Sg. (B) Schematic representation of a portion of the N-glycan biosynthetic pathway showing the reactions catalyzed by MGAT1, MAN2A2, and MGAT2 that precede the generation of branched complex N-glycans and the consequences of deleting Mgat1 in Sg (cKO) or Man2a2 globally (KO) (see text for references). Symbols for sugars are from the Symbol Nomenclature for Glycans (Neelamegham et al., 2019). (C) PCR genotyping of gDNA from enriched germ cells of 22-day Mgat2[F/F] and Mgat2[F/F]:Stra8-iCre males. * Non-specific band. (D) qRT-PCR of cDNA from 22-day Mgat2[F/F] and Mgat2 cKO germ cells to determine expression of Mgat2 and Mgat1 transcripts relative to Actb and Gapdh. (E) Western blot analysis of MGAT2 in germ cell lysates of Mgat2[F/F] and Mgat2 cKO 22-day males. The blot quantitated is shown in Supplementary Figure S1C. Histograms are mean ± SEM. Each symbol represents an independent germ cell preparation. p-values were determined by Student’s t-test (two-tailed, unpaired) *p < 0.05, **p < 0.01.

FIGURE 2

Mgat2 cKO germ cells lack MGAT2 and complex N-glycans. (A–C) Immunohistochemistry of MGAT2 expressed in 7-week Mgat2[F/F] and Mgat2[F/F]:Stra8-iCre testis sections (representative of n = 3 mice per group, two sections per mouse). (D–G) Lectin histochemistry with L-PHA and GSA-II in 7-week Mgat2[F/F] and Mgat2[F/F]:Stra8-iCre testis sections shows lectin binding to complex N-glycans only in Mgat2[F/F] sections (representative of n = 3 mice per group, two sections per mouse). Scale bars 20 µm. Related low-power images are shown in Supplementary Figure S2. Note that tubule diameters of Mgat2 cKO testes are smaller than Mgat2[F/F] controls.

FIGURE 3

Mgat2 cKO males do not make sperm. A–D, representative testis sections from 7-week Mgat2[F/F] and Mgat2[F/F]:Stra8-iCre males stained with H&E (n = 3–4 mice per group, two sections per mouse). An MNC and a vacuole (V) are indicated. Scale bars (A, B) 50 μm; (C, D) 20 µm. Images from 4-week males are shown in Supplementary Figure S3, (E) Weight of both testes in 7-week Mgat2[F/F] and Mgat2 cKO mice. (F) Testis to body weight ratio in the same mice. (G) Testis tubule diameter in the same mice. Thirty tubules from one section were measured per mouse. (H–K) Representative epididymis sections from 7-week Mgat2[F/F] and Mgat2 cKO males stained with H&E (n = 3–4 mice per group, two sections per mouse). Scale bars (H, I) 50 μm; (J, K) 20 µm. Images of epididymides from 4-week males are shown in Supplementary Figure S3, (L) qRT-PCR for marker genes in cDNA of testis cell types from 22-day Mgat2[F/F] or Mgat2 cKO germ cells. Histograms are mean ± SEM. Each symbol represents an individual mouse. Significance was determined by Student’s t-test (two-tailed, unpaired *p < 0.05 **p < 0.01, ****p < 0.001) or one-tailed, unpaired, (*) p < 0.05.

FIGURE 4

Testis morphology in prepubertal Mgat2 cKO mice. (A) Testes weight in 22-day Mgat2[F/F] and Mgat2 cKO male mice. (B) Testes to body weight ratio in the same mice. (C) Germ cell (GC) protein to testis weight (TW) ratio in 22-day Mgat2[F/F] and Mgat2 cKO males. (D, E) Representative 22-day testis sections stained with H&E. Scale bars 50 µm. (F, G) Morphology of single tubules stained with H&E. Scale bars 20 µm. (H) Testes weight in 15-day Mgat2[F/F] and Mgat2 cKO males. (I) Testes to body weight ratio in the same mice. (J) Germ cell protein to testis weight ratio in 15-day Mgat2[F/F] and Mgat2 cKO males. (K, L) Representative 15-day testis sections stained with H&E. Scale bars 50 µm. (M, N) Morphology of single tubules stained with H&E. Scale bars 20 µm. Histograms are mean ± SEM. Each symbol represents an individual mouse. Significance was determined by Student’s t-test (two-tailed, unpaired); *p < 0.05, ****p < 0.0001.

FIGURE 5

RNA-seq of 15-day Mgat2[F/F] and Mgat2 cKO germ cell RNA. (A) Heatmap of protein-coding DEGs at Padj < 0.05. (B) Volcano plot of the same protein-coding DEGs. (C) SRPLOT dot plot of enrichment analysis for GO: Cellular Component. (D) SRPLOT dot plot of enrichment analysis for GO: Biological Process.

FIGURE 6

GSEA of protein-coding DEGs. (A) Heatmap of up- and downregulated genes in 15-day Mgat2[F/F] and Mgat2 cKO germ cells from the mouse HALLMARK collection of gene sets. (B) Top gene set determined by GSEA in the HALLMARK collection. (C–E) Top three gene sets determined by GSEA of the Gene Ontology biological process (GOBP) mouse c5 collection.

FIGURE 7

Pathway enrichment analysis by IPA. (A) Network 2 in the IPA analysis of 20,167 mapped IDs. Mgat2[F/F] vs. Mgat2 cKO germ cell DEGs using Expr log ratio, Expr fold-change, Expr p < 0.05, and Padj < 0.05. (B) Network 1 in the IPA analysis of 20,167 mapped IDs. Mgat2[F/F] vs. Mgat2 cKO germ cell DEGs using only Padj < 0.05. (C) Network 2 for the IPA analysis described in (B).

FIGURE 8

AKT and ERK signaling in Mgat2 cKO germ cells. Germ cell lysates were prepared from 22-day or 15-day control and Mgat2 cKO males. (A) Western blots of pAKT and AKT in 22-day Mgat2[F/F] or Mgat2 cKO germ cell lysates. The star designates a non-specific band consistently detected with the Ab to AKT. Histograms at right show the relative ratio of pAKT:AKT in independent lysates. (B) Western blots of phosphorylated and total ERK1 and ERK2 in Mgat2[F/F] or Mgat2 cKO germ cell lysates and histograms showing the relative ratio of pERK1/2:ERK1/2 in 22-day germ cells. (C) Western blots of phosphorylated and total ERK1 and ERK2 in Mgat2[F/F] or Mgat2 cKO germ cell lysates and histograms showing the relative ratio of pERK1/2:ERK1/2 in 15-day germ cells. Histograms are mean ± SEM. Each symbol represents the lysate from an individual male. Significance was determined by Student’s t-test (two-tailed, unpaired), *p < 0.05, **p < 0.01 or one-tailed, unpaired t-test (*) p < 0.05.