Characterization of membrane progestin receptor α (mPRα) of the medaka and role in the induction of oocyte maturation.

Oocyte maturation in medaka is induced by the maturation-inducing hormone (MIH) via its membrane receptor. The most likely candidates for the membrane receptor are membrane progestin receptors (mPRs). In order to characterize the mPRα subtype of medaka, a human cell line expressing the mPRα gene of medaka was established and its steroid binding property was assessed. The α subtype exhibited high binding affinity for 17,20ß-DHP, the MIH in medaka. Treatment with a morpholino antisense oligonucleotide to mPRα blocked oocyte maturation in vivo. These results suggest that the medaka mPRα protein acts as an intermediary during MIH-induced oocyte maturation in medaka in a manner similar to that described previously for fish species.

Generation of Transparent Zebrafish with Fluorescent Ovaries: A Living Visible Model for Reproductive Biology.

The transparent zebrafish enables researchers to study the morphology and distribution of cells and tissues in vivo. To capture the dynamic processes of germ cell proliferation and juvenile ovarian development in zebrafish in vivo, we established transgenic (TG) lines to allow us to monitor the changes in the ovaries of living fish. The original transgenic line with ovarian fluorescence was occasionally established. Although the cDNA integrated in the strain was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka ß-actin promoter, expression of EGFP is restricted to the oocytes and gills in adult fish. Mutant strains with transparent bodies, roy and ruby, were isolated in zebrafish. In this study, we crossed the TG strain with fluorescent ovary with transparent strains and established the TG (ß-actin:EGFP);ruby strain. The strain is highly transparent, and the oocytes are easily observed in living fish. We identified a fluorescent tissue that might contain the undifferentiated germ cells close to the cloaca in the strain. This strain can be used for analysis of ovarian development in vivo.

Purification of the goldfish membrane progestin receptor α (mPRα) expressed in yeast Pichia pastoris.

Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an active form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-ß-D-maltoside (DDM) from yeast membranes, the recombinant protein was purified using three different columns: first it was affinity-purified over nickel-nitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.

Induction of female-to-male sex change in adult zebrafish by aromatase inhibitor treatment.

This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

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.