Characterization of multiple membrane progestin receptor (mPR) subtypes from the goldfish ovary and their roles in the induction of oocyte maturation.

Oocyte maturation (OM) in goldfish is induced by the maturation inducing hormone (MIH) via its membrane receptor. Previously, we described the cloning of the membrane progesterone receptor alpha (mPRα or paqr7b) cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIH induction of OM in goldfish. Three mPR subtypes have been identified in fish by cDNA cloning or by in silico analysis of genome sequence databases. In order to investigate the potential roles of the mPR subtypes in oocyte maturation, we cloned additional mPRs from a goldfish ovarian cDNA library. RACE amplification, and screening of the cDNA library identified one ß (paqr8) and two γ subtypes (paqr5) (hereafter referred to as γ-1 and γ-2), respectively. Tissue distribution of mPR subtypes showed differential expression pattern. However, in addition to mPRα, the ß, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Cell lines expressing the ß, γ-1 and γ-2 genes were established and their steroid binding properties compared. The ß subtype exhibited higher binding affinity than the γ subtypes for 17,20ß-DHP, the MIH in goldfish. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRß blocked the induction of oocyte maturational competence, whereas injection of antisense oliogonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that the goldfish mPRß protein acts as an intermediary during MIH induction of OM in goldfish, in a manner similar to that described previously for mPRα.

Melatonin receptor of a reef fish with lunar-related rhythmicity: cloning and daily variations.

Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the regulation of circadian rhythms in various species. For overall understanding of 'circa' rhythms in the golden rabbitfish, Siganus guttatus, which exhibits restricted lunar-related rhythms and spawns synchronously around the first quarter moon, the aim of the present study was to clone a melatonin receptor (Mel(lb)) cDNA and examine daily variations of Mel(lb) mRNA expression in certain tissues of the rabbitfish. The full-length Mel(lb) cDNA (1808 bp) contained an open reading frame to encode a protein with a length of 354 amino acids, which was highly homologous to a protein of nonmammalian species. Northern blot analysis showed transcripts of Mel(lb) in the brain and retina. Real-time quantitative polymerase chain reaction analysis also revealed expression of Mel(lb) in all tissues tested. Significantly high expression of the gene during daytime was evident in the liver and kidney. When the expression of Mel(lb) was examined in the brain and retina under conditions of light/dark cycles or constant darkness, daily and circadian variations of gene expression with two increases during daytime and nighttime for the brain and a single increase during nighttime for the retina were recognized. Moreover, daily variations in the expression of Mel(lb) were observed in the cultured pineal gland. These results suggest that the melatonin receptor plays a role in integration of melatonin actions in various tissues and that daily variations of Mel(lb) in the neural tissues may be related to regulation of circadian clock.