Endocrine Regulation of Maturation and Sex Change in Groupers.

Groupers are widely distributed in tropical and subtropical areas worldwide, are key species to coastal ecosystems, and valuable fishery targets. To facilitate artificial seed production technology for grouper aquaculture, the mechanisms of reproduction and gonad development are being elucidated for these important species. In addition, since groupers are sexually dimorphic fish with female-first maturity (protogynous hermaphrodite fish), research is being conducted to clarify the ecological mechanism of sex change and their reproductive physiology, focusing on the endocrine system. In recent years, research on groupers has also been conducted to understand changes in the coastal environment caused by ocean warming and man-made chemicals. However, due to difficulties associated with conducting research using wild populations for breeding experiments, knowledge of the physiology and ecology of these fish is lacking, especially their reproductive physiology. In this review, we present information on the reproductive physiology and endocrinology of groupers obtained to date, together with the characteristics of their life history.

Male Pheromones Induce Ovulation in Female Honeycomb Groupers (Epinephelus merra): A Comprehensive Study of Spawning Aggregation Behavior and Ovarian Development.

This study characterizes the spawning phenomena of the honeycomb grouper (Epinephelus merra), which is a lunar-synchronized spawner that spawns a few days after full moon. To elucidate the aggregation characteristics of wild honeycomb groupers, the numbers of males and females at the spawning grounds were counted before and after the full moon. Approximately 20 males were consistently observed at the spawning grounds throughout the study period. Females appeared several days after full moon and rapidly increased in number, peaking four days after full moon (41 individuals). The maturation status of the females aggregating at the spawning grounds was investigated. The gonadosomatic index increased rapidly three days after full moon, and ovulation was confirmed. Individuals with ovulatory eggs were present for three days, after which the number of females at the spawning grounds decreased. Additionally, the role of males in final oocyte maturation (FOM) and ovulation in females during the spawning phase was investigated in captivity. FOM was induced in females reared in water with mature males, suggesting that male pheromones in the water induced FOM via activation of the hypothalamic-pituitary-gonadal axis. This suggests that spawning at the natural spawning grounds was the result of male-female interactions via pheromones.

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction.

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Expression profiles of hepatic vitellogenin and gonadal zona pellucida subtypes in gray mullet (Mugil cephalus) with 17α-ethinylestradiol-induced gonadal abnormality.

The subtypes of zona pellucida (zp), primarily expressed in female gonads, are considered novel molecular markers for testis-ova (or intersex), a type of gonadal abnormality caused by environmental estrogens (EEs) in Japanese medaka (Oryzias latipes). However, the association between testis-ova and the expression of gonadal zp subtypes is unclear in other teleost species, particularly in species studied in field surveys. In this study, 17α-ethinylestradiol (EE2) was orally administrated at 4-4000 ng/g body weight (BW)/day for 28 days to gray mullets (Mugil cephalus), and gonadal abnormalities were studied using histological analysis. The expression profiles of gonadal zp subtypes (zpb and zpc5) were analyzed to evaluate their suitability as gonadal abnormality markers by comparing with a hepatic vitellogenin (vtg) subtype (vtgAb). The oral administration of EE2 40 and 400 ng/g BW/day for 28 days induced significant gonadal zpb expression, and the gonads showed moderate abnormality (testis-ova). Conversely, the gonadal zpc5 levels decreased significantly in response to the oral administration of EE2 at 4000 ng/g BW/day for 28 days, and the gonads exhibited severe abnormalities. The hepatic vtgAb levels increased upon EE2 treatment regardless of gonadal abnormality. Therefore, the gonadal zpb levels and hepatic vtgAb levels served as appropriate markers for testis-ova and EE2 presence, respectively. However, the diagnosis of severe gonadal abnormality using gonadal zpc5 was moderately accurate. The findings suggest that the combination of vtgAb, zpb, and zpc5 is a potential marker for gonadal abnormality caused by EE contamination in gray mullet. That said, the potential of zpc5 should be reconsidered to determine if it shows greater accuracy in a larger or more diverse population.

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.

Expression profile of GnRH-like peptide during gonadal sex differentiation in the cephalopod kisslip cuttlefish, Sepia lycidas.

Gonadotropin-releasing hormone (GnRH) is one of the most important neuroendocrine regulators for animal reproduction. GnRH-like peptide (GnRH-like) has recently been shown to play a critical reproductive role mainly in gametogenesis or steroidogenesis in the gonads of some molluscs, including cephalopods. However, its involvement in gonadal sex differentiation remains unknown. Here, we show the expression profile of GnRH-like in the brain of the cephalopod kisslip cuttlefish, Sepia lycidas, throughout gonadal sex differentiation, by quantitative real time RT-PCR and immunohistochemistry. We found that GnRH-like could be detected in the brain at a sexually undifferentiated stage, and its expression level significantly increased upon initiation of gonadal sex differentiation. However, no significant difference in GnRH-like expression levels was observed between sexes during gonadal sex differentiation. Additionally, we demonstrated immunoreactivity of GnRH-like in glial cells or immature neurons, which are mainly distributed in the non-reproductive related area of the cephalopod brain, suggesting the immature function of the reproductive endocrine axis during early ontogenesis. Our results demonstrate for the first time, the expression profile of GnRH-like during early ontogenesis in cephalopods.

Gonadal sex differentiation and development during early ontogenesis in the breeding kisslip cuttlefish (Sepia lycidas).

To understand and obtain basic information on sex differentiation in the kisslip cuttlefish (Sepia lycidas), the gonadal sex differentiation process was investigated histologically. An undifferentiated gonad consisting of germ cells and somatic cells was found to form at a caudal site in the space between the internal yolk sacks of cuttlefish embryos at 14 and 21 days after spawning (DAS). Sexual dimorphism in the gonad was first detected at around 28 DAS. Meiotic oocytes were observed as the first visible morphological characteristic of ovaries in the gonads of some cuttlefish embryos at 28 DAS. In other individuals, neither meiotic germ cells, nor the appearance of a testicular structure, were observed in the gonad even after 10 days post hatching (DPH). Seminiferous tubules, consisting of a small number of spermatogonia and a surrounding basement membrane, were the first visible morphological characteristic of the testis in the male gonad, detected at around 20 DPH. This is the third report on the gonadal sex differentiation process in cephalopods.

Solid-state AIEnh-circularly polarised luminescence of chiral perylene diimide fluorophores.

Solid-state organic fluorescent materials are important for the development of electroluminescent sensing devices. Herein, we report that N,N'-bis((R)-1-phenylethyl)perylene-3,4,9,10-tetracarboxylic diimide [(R,R)-BPP] and its antipode [(S,S)-BPP], which contain extended π-electrons through planar perylenes, emit solid-state aggregation-induced-enhanced (AIEnh) circularly polarised luminescence (CPL) in inorganic (KBr) pellets and organic-polymer-film (PMMA- and myo-IPU-film) states; this CPL is difficult to observe in solution. These chiral perylene fluorophores emit AIEnh-CPL with high dissymmetry factors (g CPL) (up to 2.4 × 10-3) and high quantum yields (Φ F, up to 0.43) in the three solid matrices.

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.

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus.

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.

The role of pituitary gonadotropins in gonadal sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus.

The aim of this study was to clarify the roles of 2 gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus. To do this, the mRNA expression patterns of GTH subunits (cga, fshb, and lhb) in the fish pituitary throughout gonadal sex differentiation were investigated. Real-time reverse transcriptase (RT)-PCR showed that cga and fshb were present in the undifferentiated and ovarian differentiation stages, and that the expression levels significantly increased after ovarian differentiation (AOD). However, lhb was not expressed before ovarian differentiation (BOD) and was first detected AOD. Next, to investigate the differentiation and distribution of Fshb and Lhb-producing cells in the pituitary of fish throughout gonadal sex differentiation, immunohistochemical analysis was used to detect teleost GTH subunits. Positive immunoreactivity against Fshb and Lhb was not detected in the pituitary BOD; Fshb and Lhb-positive cells first appeared in the pituitary AOD. It therefore seems unlikely that pituitary gonadotropins play a major role in the control of gonadal sex differentiation in the Malabar grouper.

Differentiation of steroid-producing cells during ovarian differentiation in the protogynous Malabar grouper, Epinephelus malabaricus.

To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.

Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for L-lysine production.

A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP(+) to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis.