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.

Development of a giant grouper Luteinizing Hormone (LH) Enzyme-Linked Immunosorbent Assay (ELISA) and its use towards understanding sexual development in grouper.

A recombinant giant grouper Luteinizing Hormone (LH) consisting of tethered beta and alpha subunits was produced in a yeast expression system. The giant grouper LH ß-subunit was also produced and administered to rabbits for antibody development. The recombinant LH and its antibody were used to develop an Enzyme Linked Immunosorbent Assay (ELISA). This ELISA enabled detection of plasma LH levels in groupers at a sensitivity between 391 pg/ml and 200 ng/ml. Different species of grouper were assayed with this ELISA in conjunction with gonadal histology and body condition data to identify links between circulating LH levels and sexual development. We found that circulating levels of LH decreased when oocytes began to degenerate, and sex-transition gonadal characteristics were apparent when LH levels decreased further. When circulating LH levels were related to body condition (body weight/ body length), transitioning-stage fish had relatively high body condition but low plasma LH levels. This observation was similar across multiple grouper species and indicates that plasma LH levels combined with body condition may be a marker for early male identification in the protogynous hermaphrodite groupers.