(255) Investigation of Novel Ligand-Receptor Systems Controlling Kisspeptin Neurons and Gonadotropin-Releasing Hormone Neurons in Domestic Animals

Xiao Jia¹; Yukina Oshimo¹; Yoko Nakadaira¹; Fumie Magata¹; Fuko Matsuda¹
1. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

Abstract Text:

Pulsatile gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus induces gonadotropin (luteinizing hormone, LH and follicle-stimulating hormone, FSH) pulses from the pituitary, which stimulate folliculogenesis and steroidogenesis in the ovary. A high concentration of estrogen induces a surge-mode secretion of GnRH, which triggers an LH surge and then ovulation. Kisspeptin is a potent inducer of GnRH secretion and therefore an up-regulator of GnRH neurons. Kisspeptin neurons in the arcuate nucleus (ARC) are thought to be the GnRH pulse generator that further stimulates follicular development and directly receive estrogen negative feedback. Kisspeptin neurons in the preoptic area (POA) or anteroventral periventricular nucleus (AVPV) are thought to be the GnRH surge generator that further induce ovulation and directly receive estrogen positive feedback. But it is yet to be known how ARC and POA/AVPV kisspeptin neurons are oppositely regulated by estrogen and induce GnRH/LH pulses and surge, respectively.

Recently, we immortalized hypothalamic cells derived from the goat, a useful ruminant model, and established an ARC kisspeptin neuron cell line (GA28), a POA kisspeptin neuron cell line (GP64), and GnRH neuron cell lines (GP11, GP31). In the present study, we performed transcriptome analyses of these goat cell lines to discover novel factors that play an important role in GnRH pulse and surge generation in domestic animals.

GA28 and GP64 cells were cultured for 48 hours and total RNA was collected for RNA sequencing (RNA-seq). RNA-seq was performed on samples from three independent experiments (n = 3). Gene expression profiles were compared to determine differentially expressed genes (DEGs) between GA28 and GP64 cells. DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. RNA-seq was also performed on GP11 and GP31 cells (n = 3).
As a result of RNA-seq in GA28 and GP64 cells, 11,726 genes were expressed in both cells, while 421 and 497 genes were expressed only in GA28 and GP64 cells, respectively. There were 1,622 DEGs between GA28 and GP64 cells, of which 699 genes were significantly highly expressed in GA28 cells and 923 genes were significantly highly expressed in GP64 cells. GO analysis revealed that the DEGs were enriched in pathways related to immune system process, receptor regulator activity, and receptor ligand activity. KEGG analysis showed that the DEGs were enriched in neuroactive ligand-receptor interaction and cytokine-cytokine receptor interaction pathways. Ligands, including growth factors and chemokines, and their receptors, not previously reported to be expressed in kisspeptin neurons of any mammalian species, were identified in GA28 and GP64 cells. In addition, some receptors for the ligands were expressed in GP11 and GP31 cells. These results suggest that the newly found ligands and receptors may regulate ARC kisspeptin neurons, POA kisspeptin neurons, and/or GnRH neurons: they may be specifically involved in the estrogen negative/positive feedback system and/or GnRH pulse/surge generation, and they may be the regulator of GnRH neurons secreted from kisspeptin neurons. Further research is needed to confirm the actual function of these factors in regulating a center for reproductive function in domestic animals.