Phoenixin-20 Stimulates mRNAs Encoding Hypothalamo-Pituitary-Gonadal Hormones, is Pro-Vitellogenic, and Promotes Oocyte Maturation in Zebrafish

Abstract

Phoenixin-20 (PNX-20) is a bioactive peptide with hormone-like actions in vertebrates. In mammals, PNX stimulates hypothalamo-pituitary-gonadal hormones and regulate reproductive processes. Our immunohisto/cytochemical studies show PNX-like and the putative PNX receptor, SREB3-like immunoreactivity in the gonads of zebrafish, and in zebrafish liver (ZFL) cells. Intraperitoneal injection of zebrafish PNX-20 upregulates mRNAs encoding both salmon gonadotropin-releasing hormone (GnRH), and chicken GnRH-II and kisspeptin and its receptor in zebrafish hypothalamus. Similarly, luteinizing hormone receptor mRNA expression in the testis, follicle-stimulating hormone receptor in the ovary, and the kisspeptin system were upregulated in the gonads of PNX-20 injected fish. We also observed the upregulation of genes involved in the sex steroidogenic pathway (cyp11a1, cyp17a1, 17βhsd, cyp19a1a) in the gonads of PNX-20 administered fish. PNX-20 upregulates the expression of vitellogenin isoforms and estrogen receptor (esr2a and 2b) mRNAs in ZFL cells in vitro. Meanwhile, siRNA-mediated knockdown of PNX-20 resulted in the downregulation of all vitellogenin transcripts, further suggesting its possible role in vitellogenesis. PNX-20 treatment resulted in a significant increase in germinal vesicle breakdown in zebrafish follicles in vitro. Collectively, these results provide strong evidence for PNX-20 effects on the HPG axis and liver to promote reproduction in zebrafish. Advances in computational biology, especially in the use of bioinformatics tools, helped in the discovery of many regulatory molecules with hormone-like actions. A recent example is phoenixin-20 (PNX-20), which was originally identified as a reproductive regulatory peptide 1. Phoenixin was first isolated from the rat hypothalamus and bovine heart by two independent research groups 1,2. The mature phoenixin peptide is produced from an uncharacterized protein called small integral membrane protein 20 (SMIM20) or chromosome 4 open reading frame 52 (C4orf52). Phoenixin is reported to be present in two active forms, a 20 amino acid peptide named PNX-20 and a 14 amino acid one called PNX-14 1,2. PNX-14 is identical in humans, rats, mice, pigs, and dogs. PNX-20 differs in one amino acid between the coding regions of human, canine and porcine sequences 1. It was reported that PNX is expressed in various parts of the brain and peripheral tissues in mammals, with the highest expression in the hypothalamus. Treen et al., reported that a putative receptor of PNX is the G protein-coupled receptor 173 (GPR173), which is also called super conserved receptor expressed in brain 3 (SREB3) 3. PNX stimulates reproductive functions via acting on the HPG axis 1. Later it was reported that PNX mediates its reproductive regulatory effects through GPR173, and cAMP-PKA dependent pathway, acting on both gonadotropin-releasing hormone (GnRH) and kisspeptin 1 (Kiss1) expressing neurons 3. In mice, PNX positively influences Kiss1 transcription and GnRH mediated gonadotropin release 1,4. Intrac...

Characterization of the G protein-coupled receptor family SREB across fish evolution

“…Spatial patterns within the ovary suggest both oocyte-derived and follicle cell expression, which largely mirrored sreb1 and sreb3a in zebrafish, Atlantic cod, and human ovary 35 , 40 , 44 , 66 . Sreb3b expression within follicle cells may function similarly to sreb3a , which regulates cell proliferation and follicular progression through vitellogenesis and maturation 35 , 40 . In contrast, sreb -related oocyte expression may be important as stored maternal transcripts for early embryonic development.…”

“…Teleosts, however, were instead characterized by elevated testicular expression for most receptors, regardless of reproductive strategy. SREB roles in testes are poorly understood, but testicular sreb3a was detected in zebrafish somatic support cells, and PNX induces upregulation of steroidogenesis-related expression and dmrt1 , which is critical in male development 40 , 74 . As such, elevated sreb during early testicular stages in both sailfin molly and puffer likely reflect effects in promoting testicular development.…”

“…In addition, to conduct a preliminary study on spatial sreb3b expression patterns, a second ovary sample from each female was immediately removed for in situ hybridizations. Ovaries were chosen to enable comparisons between the novel sreb3b and prior work on ovarian sreb3a in both fish and mammals 35 , 40 .…”

<p>Phoenixin 14 Inhibits High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Experimental Mice</p>

“…Starting at the age of 10 weeks, the mice were fed with either NC (10% of kilocalories fat) or HFD (60% of kilocalories fat; Product D12492, Research Diets, New Brunswick, United States). 13,14 At the same time, two groups of mice were given phoenixin 14 (100 ng/g body weight) daily by gastrogavage for a duration of ten weeks. At the end of the experiment, the mice were sacrificed and the body weights were recorded.…”

“…Further, phoenixin has been reported to stimulate the expression of genes responsible for glycolysis in cultured zebrafish liver cells. 14 The liver plays a major role in the control of glucose metabolism, and it is likely that the administration of phoenixin 14 could have an influence on glucose metabolism in the liver. Its connection to the insulin pathway is another interesting topic to pursue.…”