Homeodomain Proteins SIX3 and SIX6 Regulate Gonadotrope-specific Genes During Pituitary Development.

Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.

FOXO1 transcription factor inhibits luteinizing hormone ß gene expression in pituitary gonadotrope cells.

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH ß-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in LßT2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues.

RhinoVax is an efficient adjuvant in oral immunisation of young chickens and cholera toxin B is an effective oral primer in subcutaneous immunisation with Freund's incomplete adjuvant.

Forty-five approximately 50% in-bred 14-day-old White Leghorn female chickens (Gallus domesticus) originating from 11 hens were distributed into 5 treatment groups containing one sister in each treatment group. Phase I involved oral administration of an antigen, Bovine Serum Albumin (BSA), in combination with various adjuvant preparations, either Cholera Toxin B-subunit (CTB) and/or RhinoVax (RV). A positive control group received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection. All chickens responded with immunospecific IgA, IgM and IgG antibodies in their circulation. Classical parenteral immunisation with FIA was generally the most potent mode of antigen administration. The highest immunospecific IgG concentrations recorded in the orally-immunised chickens were in the group immunised with 20% RV as the adjuvant. The concentration in this group was approximately 5 times lower than that recorded in the FIA group. For practical egg yolk polyclonal antibody production purposes, the oral regime using 20% RV as adjuvant seems an attractive alternative to the more invasive technique of injecting the antigen in FIA emulsions. In Phase 2 all chickens were subjected to traditional subcutaneous immunisation with a new antigen, human IgG emulsified in FIA. The two groups of chickens that had received CTB orally during Phase I responded with significantly higher immunospecific antibody concentrations than did the other chickens, indicating that oral administration of CTB prior to traditional parenteral immunisation may have a priming effect on the humoral immune system. The immunospecific antibody response varied between the 11 families of chickens. There was no correlation between familial responsiveness to oral and subcutaneous immunisations. Families that were high responders to oral immunisation were not high responders to parenteral immunisation and vice versa.