Haploinsufficiency of SIX3 Abolishes Male Reproductive Behavior Through Disrupted Olfactory Development, and Impairs Female Fertility Through Disrupted GnRH Neuron Migration

Abstract

Mating behavior in males and females is dependent on olfactory cues processed through both the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Signaling through the MOE is critical for the initiation of male mating behavior, and the loss of MOE signaling severely compromises this comportment. Here, we demonstrate that dosage of the homeodomain gene Six3 affects the degree of development of MOE but not the VNO. Anomalous MOE development in Six3 heterozygote mice leads to hyposmia, specifically disrupting male mounting behavior by impairing detection of volatile female estrus pheromones. Six3 is highly expressed in the MOE, main olfactory bulb (MOB), and hypothalamus; all regions essential in the proper migration of the gonadotropin-releasing hormone (GnRH) neurons, a key reproductive neuronal population that migrates along olfactory axons from the developing nose into the brain. Interestingly, we find that the reduction in Six3 expression in Six3 heterozygote mice compromises development of the MOE and MOB, resulting in mis-migration of GnRH neurons due to improper olfactory axon targeting. This reduction in the hypothalamic GnRH neuron population, by 45% in adulthood, leads to female subfertility, but does not impact male hormone levels, suggesting that male infertility is not related to GnRH neuron numbers, but exclusively linked to abnormal olfaction. We here determine that Six3 is haploinsufficient for MOE development, GnRH neuron migration, and fertility, and represents a novel candidate gene for Kallmann syndrome, a form of inherited infertility.

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

“…In agreement with others, we found fewer CRE, than LacZ, expressing cells in the adult septum. This transient expression of CRE has previously been noted [10, 45], and suggests some GnRH neurons might silence the Gnrh -promoter prior to adulthood. However, it should be noted, that once the Cre -allele has been expressed in a cell, allowing the recombination of the “flox” sequences, this DNA recombination is permanent.…”

“…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.…”

Time‐restricted feeding improves the reproductive function of female mice via liver fibroblast growth factor 21

“…37 To assess the function of the hypothalamicpituitary-gonadal axis in female mice after 22 weeks of TRF treatments, we performed a series of hormone challenge tests as previously described. 23,31,38 Intraperitoneal (IP) GnRH injections elicited a robust rise in LH in the four groups of mice ( Figure 3B), which excluded a pituitary defect. Furthermore, IP Kisspeptin (Kp-10) injections also induced LH secretion ( Figure 3C) in all groups, suggesting that GnRH neurons are present and can respond to stimulation.…”

Haploinsufficiency of Homeodomain Proteins Six3, Vax1, and Otx2 Causes Subfertility in Mice via Distinct Mechanisms

“…Compound heterozygosity is particularly hard to detect, as it requires identification of mutations in 2 distinct genes. Despite the difficulty in detecting polygenic IHH, haploinsufficiencies adversely affecting fertility have been reported in both rodents and humans [3–8].…”