The Methylcytosine Dioxygenase Ten-Eleven Translocase-2 (tet2) Enables Elevated GnRH Gene Expression and Maintenance of Male Reproductive Function

Abstract

Reproduction depends on the establishment and maintenance of elevated GnRH neurosecretion. The elevation of primate GnRH release is accompanied by epigenetic changes. Specifically, cytosine residues within the GnRH gene promoter are actively demethylated, whereas GnRH mRNA levels and peptide release rise. Whether active DNA demethylation has an impact on GnRH neuron development and consequently reproductive function remains unknown. In this study, we investigated whether ten-eleven translocation (tet) enzymes, which initiate the process of active DNA demethylation, influence neuronal function and reproduction. We found that tet2 expression increases with age in the developing mouse preoptic area-hypothalamus and is substantially higher in a mature (GT1-7) than an immature (GN11) GnRH cell line. GnRH mRNA levels and mean GnRH peptide release elevated after overexpression of tet2 in GN11 cells, whereas CRISPR/cas9-mediated knockdown of tet2 in GT1-7 cells led to a significant decline in GnRH expression. Manipulations of tet2 expression altered tet2 genome binding and histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Mice with selective disruption of tet2 in GnRH neurons (GnRH-specific tet2 knockout mice) exhibited no sign of altered pubertal timing in either sex, although plasma LH levels were significantly lower, and fecundity was altered specifically in adult male GnRH-specific tet2 knockout animals, indicating that tet2 may participate in the maintenance GnRH neuronal function. Exposure to bisphenol A, an environmental contaminant that alters GnRH neuron activity, caused a shift in tet2 subcellular localization and a decrease in histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Finally, evaluation of tet2 protein interactions in GT1-7 cells suggests that the influence of tet2 on neuronal function are not limited to nuclear mechanisms but could depend on mitochondrial function, and RNA metabolism. Together, these studies implicate tet2 in the maintenance of GnRH neuronal function and neuroendocrine control of male reproduction.

Figure 1.

Tet enzyme expression in GnRH cell lines. Comparison of tet enzyme expression in undifferentiated (GN11) and differentiated (GT1–7) GnRH cell lines. Data are presented as mean ± SEM; t tests compared means between cell lines for each tet enzyme; *, P = .02.

Figure 2.

Tet2 expression in the medial basal hypothalamus. Comparison of tet2 expression in male and female medial basal hypothalamus on p15 and p25. Data are presented as mean ± SEM; t tests compared means between postnatal days for each sex; **, P = .007; *, P = .047. No sex differences in tet2 expression were observed (two-way ANOVA, sex F_1,16 = 0.0112, P = .9171; age F_1,16 = 10.33, P = .0054, Tukey post hoc test at α = 0.05: p15 female < p25 female).

Figure 3.

Tet2 drives GnRH gene expression and peptide release in undifferentiated GnRH neurons while maintaining elevated GnRH mRNA levels in differentiated GnRH neurons. A, Comparison of tet2 mRNA in undifferentiated (GN11), differentiated (GT1–7), GT1–7 cells with disrupted tet2 expression (GT1–7(−)tet2), and GN11 cells overexpressing tet2 (GN11+tet2). Data are presented as mean ± SEM, ANOVA followed by Tukey post hoc, bars with same letter are statistically equivalent; P < .001. A, inset, Western blotting indicating differences in tet2 protein levels between respective cell lines. B, Comparison of GnRH mRNA in GN11, and GN11+tet2 cell lines (t test, P = .034). C, Comparison of GnRH mRNA in GT1–7, and GT1–7(−)tet2 cell lines (t test, P < .001). D, Measurements of average GnRH peptide release from GN11 and GN11 (+) tet2. Data are presented as mean ± SEM; t test compares means; *, P = .043.

Figure 4.

Tet2 binding and chromatin modifications across the mouse GnRH gene. A, Schematic diagram (adapted from Iyer et al [3]) of the mouse GnRH gene depicting locations of enhancers E3, E2, E1, and the GnRH promoter (P). Opposing arrows show ChIP assay primer binding locations. For reference, fold enrichment was also measured at the insulin promoter (not depicted). B–D, Fixed chromatin from GN11, GT1–7, and GN11+tet2 and GT1–7(−)tet2 cell lines was sonicated then immunoprecipitated with antibodies against Tet2, H3K4me3, or H3K27me3. DNA was then analyzed by qPCR. Data are presented as fold enrichment relative to IgG (2 (IgG C_t-specific antibody C_t), mean of 4 cultures per cell line ± SEM. Regions not assigned the same letter are significantly different by Tukey post hoc; P < .05 after two-way ANOVA.

Figure 5.

Double immunohistochemical staining for Tet2 and GnRH in the POA of GnRH cell-specific tet2 knockout mice. Photomicrographs with a confocal microscope of CON brain (top panels) and GnRH-specific tet2 knockout (gTKO) brain (bottom panels) using antibodies specific to GnRH (green, left panels) and tet2 (red, middle panels) shows that Cre-mediated recombination of the tet2 gene is restricted to GnRH neurons (overlay, right panels). Blue is a nuclear staining (DAPI). In CON animals, arrows point to a GnRH neuron with immuno-positive tet2 staining in the GnRH neuron. Arrows in gTKO panels indicate a GnRH neuron, in which tet2 staining is absent. Note that adjacent non-GnRH cells are tet2 immuno-positive.

Figure 6.

Gene expression in the hypothalamus of GnRH cell-specific tet2 knockout mice. Expression of tet2 and GnRH in the MBH/POA of male and female CON and GnRH cell-specific tet2 knockout mice (gTKO) across development. A and B, Tet2 expression increased with age in both females (**, two-way ANOVA; age, F_2,33 = 8.625, P = .001) and males (***, two-way ANOVA; age, F_2,31 = 3.668, P = .0372). C and D, GnRH expression was lower in gTKO males compared with CON littermates (#, two-way ANOVA; genotype, F_1,23 = 6.798, P = .0157) with a trend toward declining GnRH expression with age across genotypes for both sexes (male: two-way ANOVA; age, F_2,23 = 3.152, P = .0617; female: two-way ANOVA, age, F_2,21 = 3.308, P = .0564). Data are presented as expression relative to 18s; mean ± SEM, n ≥ 4 animals per sex and genotype, run in triplicate for each gene and developmental age.

Figure 7.

Mice with specific ablation of tet2 activity in GnRH neurons (gTKO) exhibit typical pubertal development in both sexes and male-specific deficits in gonadotropin release and reproductive function as young adults. A, The average day of v V.O., first estrus, and preputial separation is not different between gTKO and CON littermates. B, gTKO animals exhibit normal estrous cyclicity, with the percent time spent in each phase of the estrous cycle similar to CON animals. C, Plasma LH levels at p30 are similar between gTKO and CON animals of each sex. Plasma LH levels at p60 are similar between genotypes in female but lower in gTKO compared with CON males; data are presented as mean ± SEM; t tests compared means between postnatal days for each sex; *, P = .04. D, gTKO female fecundity did not appear compromised, but male gTKO took longer than CON animals to impregnate a fertile female; data are presented as mean ± SEM; t tests compared means between genotypes for each measurement; *, P = .034.

Figure 8.

BPA exposure alters tet2 subcellular localization and the GnRH gene chromatin environment in GT1–7 cells. A, Immunofluorescence analysis of GT1–7 cells using tet2 antibody after BPA (10nM) or vehicle exposure for 2 hours. B, Quantification of tet2 immunoreactivity in 150 cells; immunoreactivity was categorized as occurring in the cytosol, the nucleus, or both compartments. C, Schematic diagram (adapted from Iyer et al [3]) of the mouse GnRH gene depicting locations of enhancers E3, E2, E1, and the GnRH promoter (P). Opposing arrows show ChIP assay primer binding locations. Fixed chromatin from GT1–7 cells exposed to either vehicle or BPA (10nM for 2 h) was sonicated then immunoprecipitated with antibodies against Tet2, H3K4me3, or H3K27me3. DNA was then analyzed by qPCR. Data are presented as fold enrichment relative to IgG (2 (IgG C_t-specific antibody C_t), mean of 3 cultures per cell line ± SEM. Regions not assigned the same letter are significantly different by Tukey post hoc; P < .05 after two-way ANOVA.

Figure 9.

Tet2-interacting proteins in GT1–7 cells. Abundance of unique peptides identified by mass spectrometry from tet2 coimmunoprecipitates. The most prominent tet2 interactions are grouped into cellular functions associated with the interacting protein.

Figure 10.

Tet2 influences accumulation and maintenance of the activating H3K4 trimethylation in immature and differentiated GnRH neurons, respectively. Top portion, Immature GnRH neuronal cell lines (GN11) express GnRH at very low levels and less tet2 than differentiated GnRH neuronal cell lines (GT1–7). Increasing tet2 expression in GN11 cells by transient transfection leads to an elevation of GnRH mRNA and H3K4me3 at the GnRH gene neuron-specific enhancer and promoter. Bottom portion, Knockdown of tet2 expression in differentiated GnRH neuronal cultures (GT1–7 cells) leads to a loss of tet2 binding near the GnRH gene promoter, loss of H3K4me3 abundance in the same region, and reduction in GnRH mRNA levels.