Heterozygous Deletion of Ventral Anterior Homeobox (Vax1) Causes Subfertility in Mice

Abstract

The known genetic causes of idiopathic hypogonadotropic hypogonadism (IHH) are often associated with the loss of GnRH neurons, leading to the disruption of the hypothalamic pituitary gonadal axis and subfertility. The majority of IHH cases have unknown origins and likely arise from compound mutations in more than one gene. Here we identify the homeodomain transcription factor ventral anterior homeobox1 (Vax1) as a potential genetic contributor to polygenic IHH. Although otherwise healthy, male and female Vax1 heterozygous (HET) mice are subfertile, indicating dosage sensitivity for the Vax1 allele. Although Vax1 mRNA is expressed in the pituitary, hypothalamus, and testis, we did not detect Vax1 mRNA in the sperm, ovary, or isolated pituitary gonadotropes. Whereas Vax1 HET females produced normal numbers of superovulated oocytes, corpora lutea numbers were reduced along with a slight increase in circulating basal LH and estrogen. The subfertility originated in the hypothalamus in which kisspeptin and GnRH transcripts were altered along with a substantial reduction of GnRH neuron number. Although the pituitary responded normally to a GnRH challenge, diestrus females had reduced LHβ and FSHβ in diestrus. Furthermore, Vax1 HET males had reduced GnRH mRNA and neuron numbers, whereas the pituitary had normal transcript levels and response to GnRH. Interestingly, the Vax1 HET males had an 88% reduction of motile sperm. Taken together, our data suggest that Vax1 HET subfertility originates in the hypothalamus by disrupting the hypothalamic-pituitary-gonadal axis. In addition, male subfertility may also be due to an unknown effect of Vax1 in the testis.

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

“…Surprisingly, unlike Diaczok et al [30], we did not observe any fertility phenotype in the Otx2:Lhrh-cre females, which have normal ovaries (not shown), suggesting correct release of LH and FSH. The maintained fertility of Otx2:Lhrh-cre females is supported by other studies showing that fewer than 34% of GnRH neurons are required for estrous cycling and fertility [32, 50]. The greater subfertility of the Otx2:Gnrh-cre females [30], as compared to the Otx2:Lhrh-cre (this study) might be the result of germline recombination in the female Otx2:Gnrh-cre oocytes, incomplete recombination of the Otx2-flox allele when using the Lhrh-cre allele, or ectopic expression of the Gnrh-cre allele.…”

“…GnRH neuron migration is halted around birth; thus, any delay in GnRH neuron migration causes a reduction of hypothalamic GnRH neurons and this has serious consequences to GnRH neuron targeting to the ME and fertility [10, 19, 30, 32, 45]. We found an increase in the number of GnRH neurons at the cribriform plate in e17.5 old Otx2:Lhrh-cre embryos.…”

“…We found an increase in the number of GnRH neurons at the cribriform plate in e17.5 old Otx2:Lhrh-cre embryos. This increase in GnRH neurons at the entrance to the brain (cribriform plate) was associated with a reduction of GnRH neurons in the brain, although the total number of GnRH neurons in these embryos was only decreased by 21%, a reduction not expected to impact fertility [19, 32, 36, 50, 51]. However, our study was not designed to address if the differential distribution of GnRH neurons in Otx2:Lhrh-cre mice was caused by GnRH neuron death, abnormal migration, or a change in proliferation.…”

“…Procedures for pubertal onset, embryo collection and fertility assessment were performed as described in detail previously [19, 32, 33]. For fertility assays, 8-week-old Otx2:Lhrh-cre mice were housed with Otx2-flox/flox mice.…”

“…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).…”