Hypothalamic Dysregulation and Infertility in Mice Lacking the Homeodomain Protein Six6

Abstract

The hypothalamus, pituitary, and gonads coordinate to direct the development and regulation of reproductive function in mammals. Control of the hypothalamic–pituitary– gonadal axis is dependent on correct migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus, followed by proper synthesis and pulsatile secretion of GnRH, functions absent in patients with hypogonadal hypogonadism. In this study, we identify sine oculis-related homeobox 6 (Six6) as a novel factor necessary for proper targeting of GnRH expression to the limited population of GnRH neurons within the adult mouse hypothalamus and demonstrate that it is required for proper reproductive function in both male and female mice. Female Six6-null mice exhibit a striking decrease in fertility, failing to progress through the estrous cycle normally, show any signs of successful ovulation, or produce litters. Although basal gonadotropin production in these mice is relatively normal, analysis of GnRH expression reveals a dramatic decrease in total GnRH neuron numbers. We show that expression of Six6 is dramatically increased during GnRH neuronal maturation and that overexpression of Six6 induces GnRH transcription in neuronal cells. Finally, we demonstrate that this induction in GnRH expression is mediated via binding of Six6 to evolutionarily conserved ATTA sites located within the GnRH proximal promoter. Together, these data indicate that Six6 plays an important role in the regulation of GnRH expression and hypothalamic control of fertility.

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

“…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.…”

“…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.…”

“…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.…”

Analysis of genome-wide structure, diversity and fine mapping of Mendelian traits in traditional and village chickens

“…Givens et al (2005) found evidence to support the role of DLX1 in regulating the GnRH promoter during development in mice. Several recent studies have associated the misexpression of GnRH with infertility in mice (Larder et al, 2011). DLX1 might therefore be associated with the rose comb phenotype and/or the subfertility linked to this trait.…”

SynCAM1, a Synaptic Adhesion Molecule, Is Expressed in Astrocytes and Contributes to erbB4 Receptor-Mediated Control of Female Sexual Development

“…For instance, mice carrying a dominant-negative form of erbB4 targeted to astrocytes have a delayed first ovulation, but exhibit normal adult reproductive capacity (7). Additional examples are mice carrying a point mutation of the erbB1 receptor (30,31) and mice lacking the Janus-activated kinase 2 in GnRH neurons (32) or lacking the homeodomain protein Six6 also in GnRH neurons (33). The most tenable explanation for the absence of complete infertility resulting from these deficiencies is the existence of redundant and compensatory circuits that become operative when one system fails to perform.…”