Loss of Inhibin Advances Follicle Activation and Female Puberty Onset but Blocks Oocyte Maturation in Zebrafish

Inhibin was first characterized in mammals as a gonadal dimeric protein that inhibited pituitary follicle-stimulating hormone (FSH) secretion. As in mammals, the inhibin-specific α subunit (INHA/Inha/inha) has also been characterized in teleosts; however, its functions and physiological importance in fish reproduction remain unknown. Using CRISPR/Cas9 method, we generated an inha-deficient zebrafish line and analyzed its reproductive performance. As expected, pituitary expression of fshb increased significantly in both the young and the adult inha mutant. The expression of lhb also increased in the mutant, but only in sexually mature adults. Interestingly, the expression of activin βA (inhbaa) increased significantly in both the ovary and the testis of inha mutant, and the expression of ovarian aromatase (cyp19a1a) also increased dramatically in the mutant ovary. The juvenile female mutant showed clear signs of early follicle activation or precocious puberty onset. However, the adult female mutant was infertile with follicles arrested at the full-grown stage without final oocyte maturation and ovulation. Although follicle growth was normal overall in the mutant, the size and distribution of yolk granules in oocytes were distinct and some follicles showed granulosa cell hypertrophy. In contrast to females, inha-null males showed normal spermatogenesis and fertility. As reported in mammals, we also found sporadic tumor formation in inha mutants. Taken together, our study not only confirmed some conserved roles of inhibin across vertebrates, such as inhibition of FSH biosynthesis and tumor formation, but also revealed novel aspects of inhibin functions such as disruption of folliculogenesis and female infertility but no obvious involvement in spermatogenesis in fish.

A267 ROLE OF INTESTINAL EPITHELIAL NUCLEAR RECEPTOR HNF4A DURING METABOLIC DISORDERS

Background HNF4α belongs to the hormone nuclear receptor family and is expressed in liver, intestinal epithelium and pancreas where it regulates genes involved in the control of metabolism. Inactivating mutations in the HNF4A gene cause several forms of maturity-onset diabetes of the young (MODY). However, the specific deletion of Hnf4a in mouse pancreatic beta cells does not lead to diabetes, suggesting the contribution of other tissues, such as the intestine, are necessary for the progression of the disease. Aims Our main hypothesis was that intestinal epithelial HNF4α regulates gene products that act through a paracrine mode of communication in the context of glucose metabolism. The aims of this study were to investigate the impact of deleting Hnf4a in the mouse intestinal epithelium during glucose homeostasis and to identify molecular mechanisms involved during glucose-induced obesity resistance. Methods The Villin-Cre recombinase transgenic mouse model was used to conditionally delete Hnf4a in the intestinal epithelium (Hnf4adeltaIEC). Hnf4adeltaIEC mice were put on a high sugar diet for 8 to 12 weeks, using a 30% sucrose supplemented ab lithium water. Blood glucose values in controls and mutants were measured from whole venous blood from fasted mice or during glucose and insulin tolerance tests. Mouse serum hormone levels (Ghrelin, Fibroblast-growth factor-15 (Fgf15), Insulin, Cholecystokinin (CCK), etc.) were measured using mouse ELISA kits. The Promethion High-Definition Room Calorimetry System was used for indirect calorimetry and metabolic studies. Results Both male and female Hnf4adeltaIEC mice displayed a metabolic resistance to develop obesity under sucrose supplementation when compared to control mice. While male mutant mice showed a resistance to obesity after only 2 weeks of treatment, female mutant mice took at least 6 weeks to display some resistance. The gut hormones ghrelin and Fgf15 were also found modified in fasted mutant mice. Female mutant mice presented a significant increase of 1.8 fold in circulating Fgf15 and an increase of 1.4 fold in circulating ghrelin. Similar changes were observed in male mutant mice. However, only male mutant mice presented an insulin resistance and an oral glucose tolerance after between 6 and 8 weeks. Brown adipose tissue (BAT) whitening was observed after 8 weeks of sucrose treatment in control obese animals, a condition that was prevented in Hnf4adeltaIEC mice. Conclusions The identification of paracrine intestinal targets for HNF4α in association with glucose metabolism will provide a better understanding of the molecular nature of tissues crosstalk in energy balance and in metabolism disorders including diabetes and obesity. Funding Agencies CIHR