Water temperature effects on embryonic development and GnRH-like expression in kisslip cuttlefish (Sepia lycidas) hinting the potential role of GnRH-like peptide.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

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.

Changes in the Hepatic Expression of Three Vitellogenin Subtype Genes During Ovarian Development in Female White-Edged Rockfish (Sebastes taczanowskii).

Viviparous fish, including white-edged rockfish (Sebastes taczanowskii), accumulate substantial yolk mass in the oocytes; however, the details of the molecular mechanisms underlying yolk formation are not yet fully understood, especially concerning multiplicity in the yolk precursor vitellogenin (Vtg). The present study aimed to reveal the hepatic transcriptional profiles of multiple vtg gene transcripts (vtgAa, vtgAb, vtgC) during the reproductive cycle in captive female white-edged rockfish reared in an aquarium under natural photo-thermal conditions. The serum estradiol-17ß concentration and the hepatic transcript levels of all vtg subtypes increased with the progress of vitellogenesis; both levels decreased at the beginning of oocyte maturation and remained low during the gestation period. Considering the similarity in the transcriptional profiles of vtg subtypes between Sebastes and Oncorhynchus, along with the differences between Sebastes and Morone, it is suggested that the transcription patterns of multiple vtg genes relate to neither their reproductive modes (viviparity versus oviparity) nor to teleost phylogeny.

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.

Hepatic estrogen-responsive genes relating to oogenesis in cutthroat trout (Oncorhynchus clarki): The transcriptional induction in primary cultured hepatocytes and the in vitro promoter transactivation in responses to estradiol-17ß.

Estradiol-17ß (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg: chgHα, chgHß, chgL; vtg: vtgAs, vtgC; esr: esr1a, esr1b, esr2a, esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a, were increased by E2 treatment (10-6 M) at 24 and 72 h post initiation (hpi), but esr1b, esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10-11-10-6 M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10-11 M for chgHα at 24 hpi, as 10-9 M for vtgC at 72 hpi, and as 10-10 M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esr1a mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10-9 M at 24 hpi to 10-7 M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg, vtg and esr1a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHα ≥ vtgAs > chgHß > chgL ≥ vtgC ≥ esr1a when mediated by Esr1a, chgHß > chgHα > chgHL > vtgAs ≥ vtgC ≥ esr1a by Esr1b, chgHß ≥ chgL > chgHα ≥ vtgAs > vtgC > esr1a by Esr2a, and chgHß ≥ chgHα ≥ vtgAs > chgL ≥ vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg, vtg and esr1a genes in the estrogen-exposed hepatocytes.

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.

Hepatic expression profiles of three subtypes of vitellogenin and estrogen receptor during vitellogenesis in cultured female yellowtail.

Estradiol-17ß (E2) promotes the transcription of vitellogenin (Vtg) via nuclear estrogen receptor (ER). Three Vtg (VtgAa, VtgAb, and VtgC) and ER subtypes (ERα, ERß1, and ERß2) have been reported in perciform fish; however, the relationship between the transcriptional regulation of Vtg and ER subtypes remains unclear. Molecular characterization was performed and the expression profiles of vtg and er subtypes were investigated to elucidate mechanisms of synthesis of vtg subtypes in yellowtail, Seriola quinqueradiata. Primary structures and promoter regions were revealed in three subtypes of vtg and er, and all the vtg subtypes and erα were presumed to be estrogen-responsive genes. When all vtg subtypes were expressed significantly in the liver, hepatic expression levels of all the er subtypes also increased. Conversely, although plasma E2 concentrations did not change significantly, the concentrations were high at the same time. Hepatic expression levels of all the vtg subtypes were highly correlated with hepatic erα, rather than with hepatic erß subtypes and plasma E2. A high positive correlation was also observed between erß1 and ß2, which seemed to be highly expressed at the pre- and late-vitellogenic stages. The results of the present study suggest that the transcription of the three vtg subtypes are regulated by three ER subtypes jointly, and ERα is the key transcription factor regulating the three vtg subtypes in yellowtail.

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.

The Mechanism of Low-Temperature Tolerance in Fish.

In this chapter, we cover the life history of fish in low-temperature environments, including their overwintering behavior and the physiological mechanisms by which they maintain life in cold environments, based on research to date. There is relatively little research on low-temperature tolerance of fish, compared with research on this phenomenon in mammals and birds, which are also vertebrates, and the mechanisms in fish have not been fully elucidated. First, we cover the life history of fish that overwinter by entering dormancy or hibernation. Next, we describe the mechanism that controls body temperature in fish that survive low-temperature environments. Finally, we introduce the physiological mechanisms for survival in extremely low-temperature environments, particularly antifreeze proteins.

Molecular cloning of vitellogenin gene promoters and in vitro and in vivo transcription profiles following estradiol-17ß administration in the cutthroat trout.

Transcription of vitellogenin (vtg) genes are initiated when estradiol-17ß (E2)-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg: vtgAs; minor C-type vtg: vtgC) by E2 was investigated under co-expression of a potential major transcriptional factor, erα1, in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC50) was similar among all vtg promoters and also to the EC50 of E2 administered to induce vtg transcription in vivo. This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression.

Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: Evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth.

To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain (cltc) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc (cltc-a1, cltc-a2, cltc-b) to be expressed in 10 different tissues including the ovary. The cltc-a1 alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctlc signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of ~170kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of cltc/Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr/Vtgr in this species, constitute the first empirical evidence that cltc-a1/Cltc-a1 is involved in Vtg endocytosis via the Vtgr in teleost fish.

Molecular cloning and partial characterization of a low-density lipoprotein receptor-related protein 13 (Lrp13) involved in vitellogenin uptake in the cutthroat trout (Oncorhynchus clarki).

Multiple ovarian membrane proteins that bind vitellogenin (Vtg) have been detected in teleosts. One of these Vtg receptors was recently identified as low-density lipoprotein receptor-related protein 13 (lrp13/Lrp13) in perciform species, but little is known about this Vtg receptor in salmonid fish. In this study, a cDNA encoding a putative Vtg receptor with 13+1 ligand binding repeats (lr13+1) was cloned from the ovary, and identified as an lrp13 ortholog for cutthroat trout (Oncorhynchus clarki). This lrp13 was predominantly expressed in the pre-vitellogenic stage ovary, and its expression decreased during vitellogenesis. Ovarian localization of Lrp13 was observed by immunohistochemistry using specific antiserum against recombinant Lrp13. Lrp13 immunoreactivity was observed at the oolemma, throughout the zona radiata, and within the perivitelline space between the zona radiata and granulosa cells in ovarian follicles at both the lipid-droplet and vitellogenic stages of growth-an expression pattern that mimics that of a lr8/LR8-type Vtg receptor in this species and of lrp13/Lrp13 in Morone species. Six discrete Vtg-binding proteins were detected in cutthroat trout ovarian membrane proteins when probing with a digoxygenin-labeled salmonid A-type Vtg (VtgAs) followed by chemiluminescent ligand detection. Western blotting using the anti-Lrp13 serum revealed a broad signal consisting of two proteins with masses ranging from ∼190 to ∼210 kDa, which corresponded with some of the VtgA-binding proteins. These findings suggest that, in addition to lr8/LR8, lrp13/Lrp13 acts as a VtgA receptor in trout.

Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins.

Fish egg yolk is largely derived from vitellogenins, which are synthesized in the liver, taken up from the maternal circulation by growing oocytes via receptor-mediated endocytosis and enzymatically processed into yolk proteins that are stored in the ooplasm. Lipid droplets are another major component of fish egg yolk, and these are mainly composed of neutral lipids that may originate from maternal plasma lipoproteins. This review aims to briefly summarize our current understanding of the molecular mechanisms underlying yolk formation in fishes. A hypothetical model of oocyte growth is proposed based on recent advances in our knowledge of fish yolk formation.

Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki).

A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95-105kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species.