Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame.

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).

In vitro and in vivo gene transfer in the cloudy catshark Scyliorhinus torazame.

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

[Multiple extramedullary plasmacytomas responding to a reduced dose of carfilzomib following drug-induced thrombotic microangiopathy].

Herein, we report the findings of a 79-year-old male patient who presented with multiple extramedullary plasmacytomas following a relapse of primary plasma cell leukemia. He developed thrombotic microangiopathy (TMA) while receiving carfilzomib, lenalidomide, and dexamethasone (KLd) therapy. He was diagnosed with plasma cell leukemia 3 years ago; he demonstrated a very good partial response (VGPR) after undergoing two regimens, including either bortezomib or lenalidomide, and he had been followed up without any other treatment due to complications of infection. Following relapse, KLd was initiated. On day 7 of KLd, TMA developed; therefore, the treatment was discontinued. The TMA improved only with the discontinuation of KLd. A reduced dose of KLd was readministered; the TMA did not relapse. He demonstrated VGPR after three courses of reduced-KLd; he has since remained in remission through ten courses. Therefore, carfilzomib therapy may be useful in relapsing and refractory cases. Drug-induced TMA has been reported to be caused by either immune-mediated or dose-dependent toxicity mechanisms. In patients who develop dose-dependent TMA with carfilzomib, dose reduction could be considered in cases showing an effective response to the treatment.

Germ cells in the teleost fish medaka have an inherent feminizing effect.

AUTHOR SUMMARY: Germ cells are the only cells that can transfer genetic materials to the next generation via the sperm or egg. However, recent analyses in teleosts revealed another essential role of germ cells: feminizing the gonads. In our study, medaka mutants in which gametogenesis was blocked at specific stages provides the novel view that the feminizing effect of germ cells occurs in parallel with other reproductive elements, such as meiosis, the sexual fate decision of germ cells, and gametogenesis. Germ cells in medaka may have a potential to feminize gonads at the moment they have developed.

Long-lasting redundant gnrh1/3 expression in GnRH neurons enabled apparent switching of paralog usage during evolution.

Expressed subtype of paralogous genes in functionally homologous cells sometimes show differences across species, the reasons for which have not been explained. The present study examined hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons in vertebrates to investigate this mechanism. These neurons express either gnrh1 or gnrh3 paralogs, depending on the species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expression of gnrh1 and gnrh3 in a single neuron in piranha and hypothesized that it may represent an ancestral GnRH system. Moreover, the gnrh1/gnrh3 enhancer of piranha induced reporter RFP/GFP co-expression in a single hypophysiotropic GnRH neuron in both zebrafish and medaka, whose GnRH neurons only express either gnrh3 or gnrh1. Thus, we propose that redundant expression of gnrh1/3 of relatively recent common ancestors may be the key to apparent switching of the paralog usage among present-day species.

Recent advances in the understanding of teleost medaka ovulation: the roles of proteases and prostaglandins.

Ovulation is the process of liberating oocytes from the preovulatory follicles, and is observed in the ovaries of virtually all female vertebrate animals. Compared with mammalian species, there have been far fewer studies that address the ovulatory mechanisms of non-mammalian species. We have examined the molecular mechanism of follicle rupture during ovulation using the teleost model, medaka, or Oryzias latipes. Follicle rupture in medaka ovulation involves the cooperation of the tissue inhibitor of metalloproteinase-2b protein with at least three matrix metalloproteinases (MMP): membrane type-1 MMP (MT1-MMP), MT2-MMP, and gelatinase A. Our studies also indicate that the serine protease, i.e., plasmin, participates in the rupture for only a few hours prior to the activation of MMP-mediated hydrolysis at ovulation. The involvement of prostaglandin E2 (PGE2) in medaka ovulation was also demonstrated. Cyclooxygenase-2 and PGE2 receptor subtype EP4b were respectively shown to be an enzyme responsible for PGE2 synthesis and a receptor for the generated ligand in the preovulatory follicles. Based on the results obtained from our studies of fish, we discuss the similarities and differences in vertebrate ovulation compared with mammalian species.

The PGE2/Ptger4b pathway regulates ovulation by inducing intracellular actin cytoskeleton rearrangement via the Rho/Rock pathway in the granulosa cells of periovulatory follicles in the teleost medaka.

We have previously shown that the prostaglandin E2/Ptger4b receptor system is involved in ovulation in teleost medaka and induces intracellular actin cytoskeleton rearrangement in the granulosa cells of preovulatory follicles. In this study, we investigated the signaling pathways through which prostaglandin E2 induces a change in the actin cytoskeleton. Treating preovulatory follicles with GW627368X (Ptger4b antagonist), a Rho inhibitor, or Y-27632 [Rho-associated protein kinase (Rock) inhibitor] inhibited not only in vitro follicle ovulation but also intracellular actin cytoskeleton rearrangement. Active Rhoa-c and Rock1 were detected in follicles immediately before ovulation. GW627368X also inhibited Rhoa-c activation and cytoskeleton rearrangement. PGE2-induced actin cytoskeleton rearrangement was not observed in the Ptger4b-, Rhoa-c-, or Rock1-deficient OLHNI-2 cells. These results indicate that the PGE2/Ptger4b pathway regulates intracellular actin cytoskeleton rearrangement via the Rho/Rock pathway in the granulosa cells of preovulatory follicles during medaka ovulation.

Expression and localization of collagen type IV alpha1 chain in medaka ovary.

A cDNA clone coding for the collagen type IV alpha1 chain was obtained from the ovary of the medaka, Oryzias latipes. The clone encoded a protein of 1639 amino acids including a putative 21-residue signal peptide, and the deduced amino acid sequence of the alpha1 chain was homologous to those of the proteins from other species. The mRNA of the collagen type IV alpha1 chain was expressed in various tissues of the adult fish. In situ hybridization analysis revealed that the alpha1 chain mRNA was localized in the follicle layer of all growing follicles. In the post-ovulatory follicle that had released its oocyte during ovulation, the alpha1 chain transcript was detected in a winding line surrounding the tissue. This localization pattern was different from that of gelatinase B, a marker gene for granulosa cells. A specific antibody was prepared for the medaka collagen type IV alpha1 chain. Immunohistochemical analysis with this antibody yielded results consistent with those obtained by in situ hybridization. These data indicate that, in the medaka ovary, collagen type IV is synthesized by theca cells and is localized in the basement membrane.

Collagen type-I alpha1 chain mRNA is expressed in the follicle cells of the medaka ovary.

Follicle rupture during ovulation is a well-regulated biological process of extracellular matrix degradation in the vertebrate ovary. Although proteolytic enzymes responsible for the rupture have recently been identified in the medaka, Oryzias latipes , the lack of knowledge about the ovarian expression and distribution of extracellular matrix components in lower vertebrates prevents the understanding of this process's molecular mechanism. To approach the problem, we cloned a cDNA coding for the medaka collagen type-I alpha1 chain and examined its mRNA expression in the fish ovary. The deduced amino acid sequence of the collagen type-I alpha1 chain was homologous to those of the proteins from other vertebrate species. The alpha1 chain mRNA was expressed in various tissues of the adult fish. In the ovary sections of mature female fish, this mRNA was detected in a line surrounding ovarian follicles of all sizes. A comparison with the distribution of gelatinase B mRNA in follicles that had just ovulated indicated that the collagen type-I alpha1 gene is expressed in the theca cells. The current results strongly suggest that collagen type I is synthesized by theca cells and is localized in the same cell layer of the follicles.

Characterization of luteinizing hormone and luteinizing hormone receptor and their indispensable role in the ovulatory process of the medaka.

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare's serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.

New evidence for the involvement of prostaglandin receptor EP4b in ovulation of the medaka, Oryzias latipes.

A cDNA for a prostaglandin E(2) (PGE(2)) receptor subtype 4, EP4b (Ptger4b), was cloned from the medaka ovary. The effect of PGE(2) was examined using COS-7 cells expressing the recombinant Ptger4b protein. An increase in intracellular cAMP levels was observed when the cells were incubated with PGE(2), but the increase in cAMP levels was nullified by the addition of the EP4 antagonist GW627368X. The expression of ptger4b mRNA was drastically induced by the addition of pregnant mare serum gonadotropin to the in vitro culture of large preovulatory follicles. In in vitro ovulation studies of the effect of GW627368X addition on follicle ovulation, the critical timing of the PGE(2)/Ptger4b interaction was suggested to be between -1 and 0 h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary.

Expression of cyclooxygenase-2 and prostaglandin receptor EP4b mRNA in the ovary of the medaka fish, Oryzias latipes: possible involvement in ovulation.

In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E(2) receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E(2) were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E(2) receptor EP4b (ptger4b) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E(2), which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E(2) in the process.