Diversity and evolution of serotonergic cells in taste buds of elasmobranchs and ancestral actinopterygian fish.

A subset of gustatory cells are serotonin immunoreactive (ir) in the mammalian taste bud. In the taste bud of lamprey, elongated gustatory-like cells are also serotonin-ir. In contrast, flattened serotonin-ir cells are located only in the basal region of the taste buds in the teleosts and amphibians. These serotonin-ir cells are termed as basal cells. To evaluate the evolution and diversity of serotonergic cells in the taste bud of amniote animals, we explored the distribution and morphology of serotonin-ir cells in the taste buds of ancestral actinopterygian fish (spotted gar, sturgeon, Polypterus senegalus) and elasmobranch (stingray). In all examined animals, the taste buds contained serotonin-ir cells in their basal part. The number of serotonin-ir basal cells in each taste bud was different between these fish species. They were highest in the stingray and decreased in the order of the Polypterus, sturgeon, and gar. While serotonin immunoreactivity was observed only in the basal cells in the taste buds of the ancestral actinopterygian fish, some elongated cells were also serotonin-ir in addition to the basal cells in the stingray taste buds. mRNA of tryptophan hydroxylase 1 (tph1), a rate-limiting enzyme of the serotonin synthesis, is expressed in both the elongated and basal cells of stingray taste buds, indicating that these cells synthesize the serotonin by themselves. These results suggest that the serotonin-ir basal cells arose from the ancestor of the cartilaginous fish, and serotonin-ir cells in the elasmobranch taste bud exhibit an intermediate aspect between the lamprey and actinopterygian fish.

Gamete-exporting organs of vertebrates: dazed and confused.

Mature gametes are transported externally for fertilization. In vertebrates, the gonads are located within the coelom. Consequently, each species has specific organs for export, which often vary according to sex. In most vertebrates, sperm ducts and oviducts develop from the Wolffian and Müllerian ducts, respectively. However, exceptions exist. Both sexes of cyclostomes, as well as females of basal teleosts, lack genital ducts but possess genital pores. In teleosts of both sexes, genital ducts are formed through the posterior extensions of gonads. These structures appear to be independent of both Wolffian and Müllerian ducts. Furthermore, the development of Wolffian and Müllerian ducts differs significantly among various vertebrates. Are these gamete-exporting organs homologous or not? A question extensively debated around the turn of the 20th century but now largely overlooked. Recent research has revealed the indispensable role of Wnt4a in genital duct development in both sexes of teleosts: zebrafish and medaka. wnt4a is an ortholog of mammalian Wnt4, which has functions in Müllerian duct formation. These results suggest a potential homology between the mammalian Müllerian ducts and genital ducts in teleosts. To investigate the homology of gamete-exporting organs in vertebrates, more detailed descriptions of their development across vertebrates, using modern cellular and genetic tools, are needed. Therefore, this review summarizes existing knowledge and unresolved questions on the structure and development of gamete-exporting organs in diverse vertebrate groups. This also underscores the need for comprehensive studies, particularly on cyclostomes, cartilaginous fishes, basal ray-finned fishes, and teleosts.

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians.

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus.

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems.

Testicular inducing steroidogenic cells trigger sex change in groupers.

Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals Possible Peripheral Regulation of the Ovary.

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -ß, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.

Shift of the Vegetal Pole Area of Full-Grown Oocytes Toward the Ovulatory Site of the Ovary in the Medaka Fish, Oryzias latipes (Beloniformes: Adrianichthyidae).

This study presents novel findings on the dynamics of growing oocytes in the ovary of the medaka fish, Oryzias latipes. In the ovary of mature females, all follicles are anchored tightly to the abdominal ovarian rete via fibrous follicular stalks on the follicle surface. The follicular stalks lie at the end of the follicle opposite the site of its attachment to the ovarian wall. Various lengths of the follicular stalks reflect the spatial arrangement of follicles in the ovary. Herein, a line that connects the follicular stalk to the opposite side of the follicle toward or attaching to the ovarian wall is called the follicle axis. The animal-vegetal axis in late stage III oocytes is already recognizable as a line that connects the center of the oocyte nucleus and the vegetal pole area; this is ascertainable by the morphological landmark of a compact distribution of granulosa cells or rudimentary attachment filaments. In growing oocytes later than stage V, the beginning of vitellogenesis, the tufted attachment filaments are located on a discrete region of the vegetal pole area. Our observations reveal that during growth, oocytes are arranged randomly between the animal-vegetal axis and the follicle axis, whereas the vegetal pole area of full-grown oocytes in preovulatory follicles turns close to the inner surface of the ovarian wall, from which mature oocytes subsequently ovulate into the ovarian lumen. It is suggested that in the O. latipes ovary, mature oocytes always transpose the vegetal pole area to the ovulatory site of the ovarian wall prior to ovulation. The expulsion of mature oocytes from the vegetal pole appears to be the regular mode of ovulation in O. latipes.

Viviparous stingrays avoid contamination of the embryonic environment through faecal accumulation mechanisms.

In viviparous (live-bearing) animals, embryos face an embryo-specific defecation issue: faecal elimination in utero can cause fatal contamination of the embryonic environment. Our data from the viviparous red stingray (Hemitrygon akajei) reveals how viviparous elasmobranchs circumvent this issue. The exit of the embryonic intestine is maintained closed until close to birth, which allows the accumulation of faeces in the embryonic body. Faecal accumulation abilities are increased by (1) the large intestine size (represents about 400-600% of an adult intestine, proportionally), and (2) the modification in the intestinal inner wall structure, specialized to increase water uptake from the faecal matter. According to the literature, faecal accumulation may occur in embryos of the lamniform white shark as well. The reproductive biology of myliobatiform stingrays and lamniform sharks is characterized by the onset of oral feeding before birth (i.e. drinking of uterine milk and eating of sibling eggs, respectively), which is expected to result in the production of large amounts of faeces during gestation. The strong ability of faecal accumulation in these lineages is therefore likely an adaptation to their unique embryonic nutrition mechanism.

Expression patterns of sex differentiation-related genes during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus.

The three-spot wrasse, Halichoeres trimaculatus, can change sex from female to male (i.e. protogyny) due to sharp decrease in endogenous estrogen. During the sex change, ovarian tissue degenerates and testicular tissue arises newly. Finally, ovarian tissue disappears completely and replaces into mature testis. In order to predict the molecular mechanisms controlling the processes of sex change, we investigated the expression patterns of four genes (rspo1, figla, sox9b and amh), which have been thought to be associated with ovarian/testicular differentiation in vertebrates. Expression levels of rspo1 and figla, which play important roles for ovarian differentiation in vertebrates, were stable until the middle stage of the sex change, and subsequently down-regulated. Therefore, it was indicated that decrease in rspo1 and figla could result from ovarian degeneration. On the other hand, basis on the expression pattern, it was indicated that sox9b and amh, which are involved in testicular differentiation in vertebrates, were implicated in testicular formation and spermatogenesis during the sex change as well. The present results could be fundamental information for investigating the relationship between these factors and E2 depletion, which is crucial trigger for sex change.

Evolution of bidirectional sex change and gonochorism in fishes of the gobiid genera Trimma, Priolepis, and Trimmatom.

Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.

Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus).

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. © 2016 Wiley Periodicals, Inc.

Basic reproductive biology of daggertooth pike conger, Muraenesox cinereus: A possible model for oogenesis in Anguilliformes.

INTRODUCTION: Eels are animals commonly used in zoological research, as these species have a unique catadromous life history and belong to a phylogenetically ancient group of Teleostei. However, eel reproduction is difficult to investigate, since mature samples are not easily obtainable in the wild. In this study, we tested daggertooth pike conger (Muraenesox cinereus), an Anguilliformes species, as a potential model for the investigation of the reproductive biology of eels. Seventy individuals were caught between June and October, which is supposed to be their spawning season, from inshore of the Seto Inland Sea. RESULTS: The lengths and ages of samples ranged from 510 to 1239 mm and three to nine years, respectively, and the sex ratio was skewed towards females (96 % of the total sample). The gonado-somatic index of the females peaked in July. Histological observation revealed that these ovaries were similar to those of other eel species and contained matured oocytes (migratory-nucleus stage), suggesting that pike conger spawn inshore in July. The plasma concentrations of sex steroid hormones (estradiol-17ß and 11-keto-testosterone) in females gradually increased during maturation and decreased after spawning, indicating the involvement of these hormones in oogenesis of pike conger. CONCLUSIONS: The present study is the first to report on characteristics of natural oogenesis in pike conger. Because naturally maturing samples can easily be captured, daggertooth pike conger may represent an excellent model for the study of reproduction in Anguilliformes.

Opposite-directional sex change in functional female protandrous anemonefish, Amphiprion clarkii: effect of aromatase inhibitor on the ovarian tissue.

INTRODUCTION: The anemonefish, Amphiprion clarkii, is a protandrous hermaphrodite. Under appropriate social conditions, male fish can become female. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the mechanism of sexual plasticity in the gonad of this fish is still unknown. To elucidate the mechanisms underlying the sexual plasticity in the ovary of female anemonefish, an aromatase inhibitor (AI, 500 µg/g diet) was administered to the functional female fish for 80 days. RESULTS: The levels of estradiol-17ß (E2) in the fish treated with AI were significantly lower than those in the control group. Three out of five fish had ambisexual gonads with active spermatogenic germ cells in the ovarian tissue. However, female fish in the AI-treated group prior to treatment and those in the control group displayed no testicular characteristics in their developed ovaries. This result strongly suggests that germ cells with bipotentiality or spermatogonial cells remain in the functional ovaries of anemonefish following sex change from functional males to functional females. There is a possibility that estrogen depletion due to AI treatment might have caused the opposite-directional sex change from functional female to male in the anemonefish. CONCLUSIONS: The anemonefish keeps their high sexual bipotential in the ovary after sex change.

Expression profile of doublesex/male abnormal-3-related transcription factor-1 during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus.

Sex change in fish involves a dramatic transformation of gonadal tissue and a switch in gametogenesis. Doublesex/male abnormal-3-related transcription factor-1 (DMRT1), encoded by the DMRT1 gene, is involved in testicular differentiation in a wide range of vertebrates as well as in sexual differentiation and gonadal sex change. In the present study, we investigated changes in the expression of dmrt1 during artificial gonadal sex change in the three-spot wrasse, Halichoeres trimaculatus, by real-time quantitative PCR and immunolocalization, using an anti-wrasse-Dmrt1 antibody that we prepared. We found that dmrt1 expression was predominantly observed in the testes, and that Dmrt1 was expressed in Sertoli cells of testes and a few granulosa cells surrounding vitellogenic oocytes of the ovary. Additionally, the upregulation of dmrt1 expression was consistent with an increase in spermatogenic cyst quantity rather than proliferation of presumptive spermatogonia, suggesting that dmrt1 is involved in the progression of spermatogenesis during sex change. Changes in the localization of Dmrt1 during gonadal sex change further implied that Sertoli cells originate from somatic cells adjacent to gonial germ cells during testicular formation in the three-spot wrasse.

High temperature-induced sterility in the female Nile tilapia, Oreochromis niloticus.

High temperature treatments induce germ cell loss in gonads of vertebrate animals, including fish. It could be a reliable source for induction of sterility if the treatments led to a permanent loss of germ cells. Here we report that heat treatment at 37 °C for 45-60 days caused a complete loss of germ cells in female Nile tilapia, Oreochromis niloticus, and that sterility was achieved in fish at all stages of their life cycle. Unlike previous observations, germ cells did not repopulate even after returning them to the water at control conditions suggesting permanent depletion of germ cells. Gonadal somatic cells immunopositive for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were clustered at one end of the germ cell depleted gonads close to the blood vessel. Serum level of testosterone, 11-ketotestosterone, and 17ß-estradiol was significantly decreased in sterile fish compared to control. Body weight of sterile fish was higher than control fish at the end of experiment. Our observations of increased growth and permanent sterilization in the high temperature-treated fish suggest that this method could be an appropriate and eco-friendly tool for inducing sterility in fish with a higher thermal tolerance.

Transient sex change in the immature Malabar grouper, Epinephelus malabaricus, androgen treatment.

To clarify the cause of sex change recovery after the withdrawal of androgen treatment, immature female Malabar grouper were fed a diet containing 17alpha-methyltestosterone (MT) at 50 µg/g for 7 mo and then a normal diet for 6 mo. The MT brought about precocious sex change from immature ovaries to mature testes with active spermatogenesis, including the development of spermatozoa, and sex change reversed soon after MT treatment withdrawal. This result indicates that precocious sex change in immature Malabar grouper with oral MT treatment is impermanent. The expression of three steroidogenic enzymes (Cyp11a, Cyp19a1a, and Cyp11b) in the gonads of the Malabar grouper were analyzed immunohistochemically at the end of the 7-mo treatment. No apparent differences were seen in the expression pattern of these enzymes between the mature testes of MT-treated fish and the immature ovaries of control fish. In addition, serum estradiol-17beta and 11-ketotestosterone levels in treated fish were the same as those in control fish. These results indicate that in the case of immature Malabar grouper MT might have little effect on endogenous steroidogenesis during precocious sex change even though it induced active spermatogenesis in the gonads of treated fish. From these results, we also concluded that MT might have little effect on the steroidogenic endocrine pathway, and this is one cause of sex change recovery after treatment withdrawal.

Potential roles of arginine-vasotocin in the regulation of aggressive behavior in the mudskipper (Periophthalmus modestus).

The hypothalamic hormones, arginine-vasotocin (VT) and isotocin (IT), play central roles in osmoregulation and in the regulation of social behaviors including aggressive behavior in many vertebrates including fish. Here, we examined whether these hormones are associated with aggressive behavior in the mudskipper (Periophthalmus modestus). The mudskipper is an amphibious fish, which lives in the brackish water of river mouths and displays unique aggressive behavior. Upon introduction to each other in an experimental tank with aquatic and terrestrial areas, a pair of males can be classified as aggressive dominant or submissive subordinate based on the frequency of their aggressive acts, which is significantly higher in dominant male. Additionally, the length of stay in terrestrial area of dominant was longer than that of the subordinate. The latter remained in aquatic area almost throughout the period of behavioral observation. The expression of brain VT mRNA was significantly higher in subordinate than in dominant, whereas neither IT mRNA expression nor plasma cortisol level differed between subordinate and dominant male. On the other hand, an intracerebroventricular injection of VT increased aggressive behaviors in mudskippers. In addition to known roles of VT in mediation of aggressive behavior, these results may shed light on the role of endogenous VT toward water migration in submissive mudskippers. The amphibious fish is a valuable experimental model to observe the relationship between effects of central VT on the osmoregulation and social behavioral regulation in vertebrates.

Estrogen favors the differentiation of ovarian tissues in the ambisexual gonads of anemonefish Amphiprion clarkii.

All undifferentiated gonads of anemonefish first differentiate into ovaries, and then testicular tissue appear among ovarian tissue, and finally form ambisexual gonads with both ovarian and testicular tissues. The role of estradiol-17beta (E2) in differentiation of ovarian cells is well conserved across phyla; however, its role in development of ambisexual gonads is poorly understood. Here we demonstrate that the E2 produced during the differentiation of ovarian cells does not allow testicular cells to differentiate in the prospective ambisexual gonad. We examined the immunolocalization of the steroidogenic enzyme cytochrome aromatase (P450(arom)), which is involved in E2 production. In the gonads, numbers of the P450(arom) -positive cells increased during ovarian differentiation. However, immunopositive cells with weak signal intensity were seen in the interstitial areas among oocytes and between oocytes and testicular tissue undergoing testicular differentiation. In contrast, P450(arom) -positive cells were not found in any testicular tissues of the ambisexual gonads. We also examined changes in E2 production in vitro in the gonads during testicular differentiation. E2 was high in the ovaries before the appearance all of testicular tissue, and decreased accompanying the differentiation of testicular tissue. These results suggest a balance of estrogen/androgen seems to be important during sex differentiation, and then a shift from estrogen to androgen production may induce testicular differentiation in the ovary. Further, exogenous E2 treatment suppressed naturally occurring differentiation of testicular cells forming exclusively ovarian tissues in the gonad in vivo, suggesting the increase of estrogen blocks the differentiation of testicular tissue and the formation of ambisexual gonad.

Epigenetic transcriptional activation of monocyte chemotactic protein 3 contributes to long-lasting neuropathic pain.

A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.