Deletion of Vax1 from Gonadotropin-Releasing Hormone (GnRH) Neurons Abolishes GnRH Expression and Leads to Hypogonadism and Infertility

Abstract

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates mammalian fertility. Herein we demonstrate a critical role for the homeodomain transcription factor ventral anterior homeobox 1 (VAX1) in GnRH neuron maturation and show that Vax1 deletion from GnRH neurons leads to complete infertility in males and females. Specifically, global Vax1 knock-out embryos had normal numbers of GnRH neurons at 13 d of gestation, but no GnRH staining was detected by embryonic day 17. To identify the role of VAX1 specifically in GnRH neuron development,Vax1(flox)mice were generated and lineage tracing performed in Vax1(flox/flox):GnRH(cre):RosaLacZ mice. This identified VAX1 as essential for maintaining expression of Gnrh1 The absence of GnRH staining in adult Vax1(flox/flox):GnRH(cre)mice led to delayed puberty, hypogonadism, and infertility. To address the mechanism by which VAX1 maintains Gnrh1 transcription, the capacity of VAX1 to regulate Gnrh1 transcription was evaluated in the GnRH cell lines GN11 and GT1-7. As determined by luciferase and electrophoretic mobility shift assays, we found VAX1 to be a direct activator of the GnRH promoter through binding to four ATTA sites in the GnRH enhancer (E1) and proximal promoter (P), and able to compete with the homeoprotein SIX6 for occupation of the identified ATTA sites in the GnRH promoter. We conclude that VAX1 is expressed in GnRH neurons where it is required for GnRH neuron expression of GnRH and maintenance of fertility in mice.

Significance statement: Infertility classified as idiopathic hypogonadotropic hypogonadism (IHH) is characterized by delayed or absent sexual maturation and low sex steroid levels due to alterations in neuroendocrine control of the hypothalamic-pituitary-gonadal axis. The incidence of IHH is 1-10 cases per 100,000 births. Although extensive efforts have been invested in identifying genes giving rise to IHH, >50% of cases have unknown genetic origins. We recently showed that haploinsufficiency of ventral anterior homeobox 1 (Vax1) leads to subfertility, making it a candidate in polygenic IHH. In this study, we investigate the mechanism by which VAX1 controls fertility finding that VAX1 is required for maintenance of Gnrh1 gene expression and deletion of Vax1 from GnRH neurons leads to complete infertility.

Keywords: GnRH; fertility; hypogonadism; transcription; ventral anterior homeobox.

Figure 1.

E17.5 Vax1 KO brains are depleted of GnRH neuron staining. Vax1 WT, HET, and KO embryos at E13.5, n = 2–5 (A–D) and E17.5 d, n = 3–4 (E–H) were processed for IHC staining for GnRH. A, E, Green boxed areas on the drawings of the sagittal mouse head sections to the left of the panels indicate where the B and F images were taken. me, Median eminence; pit, pituitary; op, olfactory placode; cp, cribriform plate. The number of GnRH-stained neurons was counted in different subregions of the brain (C, G) and in the whole brain (D, H). Crib. plate, Cribriform plate; hypo, hypothalamus. Statistical analysis by two-way ANOVA followed by Bonferroni (C, G) and one-way ANOVA compared with WT (D, H). *p < 0.05, **p < 0.01, ***p < 0.001; n = 3–4 embryos. Scale bars, 100 μm.

Figure 2.

Vax1^flox/flox:GnRH^cre mice have no GnRH-expressing neurons. A, Strategy for generating the Vax1^flox/flox:GnRH^cre mouse. The Vax1 knock-out first mouse was crossed with a flpase mouse to create the conditional allele, then that Vax1^flox mouse was crossed with a GnRH^cre mouse to create a homozygous Vax1^flox/flox with the GnRH^cre as a heterozygous allele. B, Genotyping for the floxed allele (Fl). C, IHC of GnRH in adult male Vax1^flox/flox (WT) and Vax1^flox/flox:GnRH^cre (cKO) mice. Arrow indicates where GnRH staining is expected to be at the median eminence. D, GnRH neuron lineage tracing in GnRH^cre:RosaYFP at E13.5; arrow indicates YFP expression. E, IHC of GnRH in E15.5 WT and cKO; arrows indicate GnRH neurons. Green boxed areas on the drawings of the sagittal mouse head sections to the left of the panels (D, E) indicate where the image was taken. F, Lineage tracing in adult female GnRH^cre:RosaLacZ and Vax1^flox/flox:GnRH^cre:RosaLacZ (cKO:RosaLacZ) hypothalamus. G, Comparison of GnRH^cre:RosaLacZ driven LacZ-expressing cell localization in WT and cKO:RosaLacZ mice (n = 3). Nucl acc, Nucleus accumbens; MPOA, medial preoptic area; SCN, suprachiasmatic nucleus. Scale bars, 100 μm.

Figure 3.

Vax1^flox/flox:GnRH^cre males have a micropenis and are hypogonadal. A, Image of 6-week-old WT, cHET and cKO males. Scale bar, 0.5 cm. B, Testes of adult mice. Scale bar, 0.5 cm. C, H&E staining of testes from adult WT, cHET, and cKO males. Scale bar, 10 μm. WT and cHET males have a well defined penis (white arrow), preputial separation (WT, cHET), full size testes, seminal vesicles and normal spermatogenesis, whereas cKO have a micropenis (white arrow), delayed preputial separation, very small testes and seminal vesicles, as well as an absence of spermatogenesis. D, Basal circulating LH and FSH levels. One-way ANOVA compared with control. *p < 0.05. E, Circulating LH levels after GnRH and (F) kisspeptin-10 (kiss-10) challenges. Numbers over the histogram indicate fold-change after GnRH or Kiss-10 challenge; n = 4–8.

Figure 4.

Vax1^flox/flox:GnRH^cre females have delayed pubertal onset and are hypogonadal. A, Pubertal onset was determined by vaginal opening (WT and cHET, white arrow). Panel shows images of 6-week-old females. Scale bar, 0.5 cm. B, Ovaries and uteri were collected from 25-week-old females. Scale bar, 0.5 cm. C, H&E-stained ovary. CL, Corpora lutea. Black arrows show stage 2 primordial follicles with the expected granulosa cell layer (black arrows), and some without (yellow arrows). Scale bar, 0.5 mm. D, Estrus cycling was monitored daily in adult 12- to 20-week-old WT, cHET, and cKO mice. M, Metestrus; E, estrus; P, proestrus; D, diestrus. E, Representative cell morphology for 5 d in WT and cKO females. F, Diestrus circulating LH and FSH levels. One-way ANOVA; **p < 0.01, ***p < 0.001. Circulating LH levels after (G) GnRH and (H) kisspeptin-10 (kiss-10) challenges. Numbers over the histograms indicate fold-change after GnRH or Kiss-10 challenge. n = 6–11.

Figure 5.

VAX1 regulates Gnrh1 transcription through the GnRH enhancer 1 and promoter. Levels of Vax1 mRNA were determined by A, qRT-PCR in a mature GnRH cell line (GT1-7), an immature GnRH cell line (GN11), and NIH3T3 fibroblasts (3T3). The horizontal bar indicates lowest detectable RNA level. n = 6–8. B, GT1-7 cells were transfected with siRNA containing a scrambled or Vax1 sequence. Endogenous RNA levels of Vax1 or Gnrh1 were determined by qRT-PCR. Data represent fold-change compared with scrambled siRNA; N = 3–5 in duplicates. Statistical analysis by Student's t test compared with control. *p < 0.05, **p < 0.01. C, Left, GT1-7 cells were transfected with a Vax1-expression vector (Vax1, black hatched) and its empty vector (control, black). Right, GN11 cells were transfected with Vax1-expression vector (Vax1, black hatched) and its empty vector (control, black). Data represent fold-change compared with empty vector. Statistical analysis by Student's t test compared with control. *p < 0.05, **p < 0.01. C, GN11 and GT1-7 cells were cotransfected with a luciferase reporter driven by the 5 kb promoter region of the rat GnRH gene and increasing concentrations of Vax1. All samples were transfected with equal amounts of DNA by using the empty CMV vector. Statistical analysis by one-way ANOVA followed by Tukey post hoc. *p < 0.05, ***p < 0.001. D, Cotransfection with 5′ truncations of the GnRH 5 kb promoter driving luciferase with (black) or without (white) 10 ng of Vax1. Luciferase data are expressed as fold-induction compared with pGL3. The percentage change between cotransfection with 0 versus 10 ng of Vax1 on the reporters is indicated on the graph. Statistical analysis by two-way ANOVA followed by Dunnett's multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001. E, Vax1 regulation of the RSV enhancer/promoter (RSV E/P) driving luciferase with or without the GnRH enhancer 1 (GnRH E, −1863 to −1571 bp) or GnRH promoter (GnRH P −173 to +112 bp). Data are expressed as fold-induction compared with empty vector for each reporter. Statistical analysis by Student's t test. ***p < 0.001. Luciferase transfection studies represent n = 3–5 performed in triplicate.

Figure 6.

VAX1 regulates GnRH transcription through direct binding to ATTA sites in the GnRH-E1 and proximal promoter (P). A, Deletion of potential ATTA binding sites of VAX1 within the GnRH enhancer1/promoter (GnRH-E/P or E/P). B, Cotransfection of the indicated luciferase reporters with (black) or without (white) 10 ng of Vax1 in GN11 and GT1-7 cells. Data are expressed as fold-induction compared with GnRH E/P. Statistical analysis by two-way ANOVA followed by Bonferroni. ***p < 0.001 compared with control. C, COS-1 cells were transiently transfected with a Vax1-flag expression vector or its empty vector, CMV, and harvested 24 h post-transfection. Western blot shows detection of VAX1-flag, GAPDH (glyceraldehyde-3phosphate dehydrogenase), and TBP in the nucleus (Nucl) versus the cytoplasm (Cytopl). D, Probe sequences used for EMSA covering the ATTA sites found to be involved in VAX1 regulation of Gnrh1 transcription (sites indicated in A). The underlined ATTA elements were converted to GCCG in the mutant probes (Cold mut). E, EMSA performed using nuclear extracts from COS-1 cells transfected with Vax1-flag plasmid or control expression vector. Black arrowhead indicates super shift, gray arrowhead indicates origin of supershifted band. Wild-type (Cold WT) and mutant (Cold mut) probes were used at 100-fold higher concentration than labeled WT probe.

Figure 7.

VAX1 and SIX6 compete for regulation of the Gnrh1 promoter. A, GN11 and GT1-7 cells were cotransfected with or without Six6 (as noted on figure or 200 ng) or Vax1 (as noted on figure or 20 ng). Transcriptional activity was evaluated using the GnRH-E/P-luciferase reporter or (B–D) using the 5× ctcATTAat-TK-luciferase multimer reporter. Statistical analysis was by two-way ANOVA followed by post hoc Bonferroni. Fold-changes are compared with control (0 ng Vax1, 0 ng Six6) or as indicated by bar. *p < 0.05, **p < 0.01, ***p < 0.001. Student's t test compared with control. #p < 0.05. Horizontal line indicates control level. All experiments were performed in triplicate and repeated three to six times.