Deletion ofVax1from 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 Vax1flox/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.

Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice

“…To re-validate the targeting of GnRH neurons of the 2 studied Cre -alleles [26, 27], we performed double immunohistochemistry in adult brains of Lhrh-cre and Gnrh-cre mice. First, we validated the specificity of our antibodies performing double immunohistochemistry in a wild-type mouse, where CRE is not expressed (C57BL/6J), in a CRE-expressing mouse ( Gnrh-cre+ mouse) and in a mouse model lacking GnRH expressing neurons [11]. We detected GnRH neurons in control mice (Fig.…”

“…1e, wild-type and Gnrh-cre+ mouse). This signal was specific as evidenced by the absence of GnRH staining in the negative control tissue [11] (Fig. 1e, GnRH lacking mouse).…”

“…Single immunohistochemistry and quantitative RT-PCR were performed as previously described [11, 19]. For double immunohistochemistry, the same protocol as for single immunohistochemistry was used, with the modification that after developing and imagine the anti-CRE (1/1,000) staining using Impact DAB (brown), the slides were washed in PBS-Tween 0.02%, followed by 5 min in denaturing solution (Biocare #DNS001), after which the sections were blocked in 5% goat serum and Avidin-Biotin solutions (Vector Labs), and the anti-LHRH (1/1,000) antibody was applied O/N following the regular immunohistochemistry protocol, using Impact VIP (purple) to visualize GnRH expressing cells.…”

“…All antibodies were validated using negative controls such as cre – tissue for the anti-CRE antibody, and Cre – :ROSA26-LacZ + tissue for the anti-LacZ antibody. We have previously validated the GnRH and LacZ antibodies [11, 19, 32]. The primary antibodies used for single immunohistochemistry are rabbit anti-GnRH (Thermo Scientific #PA1-121, dilution 1/1,000, RRID:AB_325077), chicken anti-beta galactosidase (Abcam #AB9361, dilution 1/1,000, RRID:AB_307210), rabbit anti-CRE recombinase (Biolegend #908001, dilution 1/500, RRID: AB_2565079), and rabbit anti-RFP (Abcam #AB62341, dilution 1/500, RRID:AB_945213).…”

“…The role of well-defined transcriptional regulatory regions, which allow specific expression of a peptide to a limited cell population, are well studied for gonadotropin-releasing hormone (GnRH or LHRH) [13-18], a neuropeptide required for pubertal onset and fertility [11, 19-22]. Despite the careful characterization of the regulatory region of Gnrh1 , it has proven difficult to create a Cre mouse that recapitulates the onset of GnRH expression and specifically targets this small neuronal population compromised of a little under 1,000 neurons in the adult mouse.…”