RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes

Abstract

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/β subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHβ subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHβ results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP⁺ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHβ subunit in pituitaries of women.

Keywords: Aging; FSH Glycoforms; Gonadotrope; N-glycosylation; Ovary; Pituitary.

Fig.1.

Hierarchical sub-cellular or pathway mapping (top to bottom) of the 52 N-glycosylation pathway enzymes identified in gonadotropes (A). Fold enrichment of genes localized to each subcellular site/organelle are shown. Fold enrichment of enzymes involved in N-glycan biosynthesis and related metabolic pathways are shown in panel B. Note that the fold enrichment refers to the number of observed genes in the gene list normalized by the total number of genes in the underlying subcellular location or pathway. Similarly, P-values were corrected for multiple testing using false discovery rate (FDR).

Fig. 2.

Fold change values of N-glycosylation pathway enzyme-encoding mRNAs determined by RNA-sequencing analysis. At least 3 pituitaries were pooled for each sample, and an N=2 was processed for FACS analysis to isolate GFP⁺ gonadotrope cells at both timepoints, 3m and 8-10m. The statistical analysis for RNA-seq data is described in Methods section 2.4. No change in expression (ratio of 1) is indicated with a blue horizontal line.

Fig. 3.

Real time qPCR analysis of expression changes in eight N-glycosylation pathway enzymes in isolated gonadotropes cells from pituitaries of 3-, 8-, and 14-month-old females. At least 4 pituitaries were pooled and processed at each timepoint, and an N=1 was used for qPCR. Values are shown as mean ± SEM. (*) indicates p < 0.05 determined by one-way ANOVA, followed by Turkey’s post-hoc test. UD= undetectable.

Fig. 4.

Serum estradiol was measured in 3-, 8-, and 14-month old OFCR females (n=5 mice) and was detectable only in mice at 3 months of age. (A). Predicted binding sites for (B) estrogen receptor −1 (ESR1) and (C) progesterone receptor (NR3C1) on eight N-glycosylation pathway enzyme-encoding genes expressed in gonadotrope cells. The sequence with the top score is shown for each gene. Data were obtained using JASPAR database analysis (Sandelin et al., 2004).