Effects of supplementation of cryopreservation media with cysteine on the post-thaw quality and fertility of brown-marbled grouper (Epinephelus fuscoguttatus) spermatozoa.

The cryopreservation process is associated with the generation of excessive reactive oxygen species, which causes a series of cellular damage to spermatozoa. The objective of the current study was to investigate the effect of different concentrations of cysteine on post-thaw sperm quality of brown-marbled grouper sperm. Semen samples were frozen with cysteine supplemented at 0.5, 1, 2, 5, 10 mM and the control group (no additive). After thawing, sperm quality parameters were analyzed. In comparison to the control, cysteine treatment groups yielded relatively higher sperm total motility, progressive motility, and curvilinear velocity. Different concentrations of cysteine had no effect on average path velocity, straight linear velocity and viability (P > 0.05), while an increase in the concentration of cysteine resulted in a significant improvement in the mitochondrial membrane potential, SOD activity, and ATP content (P < 0.05). As for lipid peroxidation, the extent of which in cysteine treated spermatozoa was less than the control, although the differences were not statistically significant (P > 0.05). In terms of fertilizing capacity, a greater hatching rate (91.7 ± 1.2%) was obtained in thawed sperm treated with 2 mM cysteine, compared to the control (84.3 ± 4.2%; P < 0.05). Overall, it is concluded that the addition of cysteine is helpful in maintaining the function of frozen-thawed brown-marbled grouper sperm, which can be recommended as an effective antioxidant to improve the semen cryopreservation efficiency.

Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides.

Spermatogonial stem cells (SSCs) are unique adult stem cells capable of self-renewal and differentiation into sperm. Grouper is a protogynous hermaphroditic fish farmed widely in the tropical and subtropical seas. In this study, we established an SSC line derived from adult testis of orange-spotted grouper, Epinephelus coioides. In the presence of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), the cells could be maintained with proliferation and self-renewal over 20 months and 120 passages under in vitro culture conditions. The cells exhibited strong alkaline phosphatase activity and the characteristics of SSCs with the expression of germ cell markers, including Vasa, Dazl, and Plzf, as well as the stem cell markers Nanog, Oct4, and Ssea1. Furthermore, the cultured cells could be induced by 11-ketotestosterone treatment to highly express the meiotic markers Rec8, Sycp3, and Dmc1, and produce some spherical cells, and even sperm-like cells with a tail. The findings of this study suggested that the cultured grouper SSC line would serve as an excellent tool to study the molecular mechanisms behind SSCs self-renewal and differentiation, meiosis during spermatogenesis, and sex reversal in hermaphroditic vertebrates. Moreover, this SSC line has great application value in grouper fish aquaculture, such as germ cell transplantation, genetic manipulation, and disease research.

Effect of chilled storage on sperm quality of basa catfish (Pangasius bocourti).

Basa catfish (Pangasius bocourti) is an indigenous species of the Lower Mekong River with increasing aquaculture value in Southeast Asia. Short-term semen storage has proven to be a valuable tool for assisted reproduction in fish, but little information is available on basa catfish. The present study was aimed at evaluating the effect of short-term semen storage on the sperm quality of basa catfish. Semen samples were kept at 4 °C for 7 days, either undiluted or diluted with Ca-F HBSS at different ratios (1:1, 1:3, and 1:6; semen:medium). Results showed that sperm quality was significantly affected by the time of chilled storage, characterized by a decline in sperm motility, viability, mitochondrial membrane potential (MMP), ATP content, and increased level of lipid peroxidation throughout the storage period. Compared with undiluted semen, diluted in Ca-F HBSS allowed better preservation of sperm quality during 7 days of chilled storage; dilution ratio at 1:1 was more effective than higher ratios (1:3 and 1:6) for prolonging sperm storability. In addition, sperm motility, viability, and ATP content decreased more rapidly than MMP, suggesting these indicators are more sensitive in detecting sperm damage of basa catfish during short-term chilled storage. These results obtained here will contribute to a better understanding of reproductive management in this species.

First Genome-wide Association Analysis for Growth Traits in the Largest Coral Reef-Dwelling Bony Fishes, the Giant Grouper (Epinephelus lanceolatus).

The giant grouper, Epinephelus lanceolatus, is the largest coral reef-dwelling bony fish species. However, despite extremely fast growth performance and the considerable economic importance in this species, its genetic regulation of growth remains unknown. Here, we performed the first genome-wide association study (GWAS) for five growth traits in 289 giant groupers using 42,323 single nucleotide polymorphisms (SNPs) obtained by genotyping-by-sequencing (GBS). We identified a total of 36 growth-related SNPs, of which 11 SNPs reached a genome-wide significance level. The phenotypic variance explained by these SNPs varied from 7.09% for body height to 18.42% for body length. Moreover, 22 quantitative trait loci (QTLs) for growth traits, including nine significant QTLs and 13 suggestive QTLs, were found on multiple chromosomes. Interestingly, the QTL (LG17: 6934451) was shared between body weight and body height, while two significant QTLs (LG7: 22596399 and LG15: 11877836) for body length were consistent with the associated regions of total length at the genome-wide suggestive level. Eight potential candidate genes close to the associated SNPs were selected for expression analysis, of which four genes (phosphatidylinositol transfer protein cytoplasmic 1, protein tyrosine phosphatase receptor type E, alpha/beta hydrolase domain-containing protein 17C, and vascular endothelial growth factor A-A) were differentially expressed and involved in metabolism, development, response stress, etc. This study improves our understanding of the complex genetic architecture of growth in the giant grouper. The results contribute to the selective breeding of grouper species and the conservation of coral reef fishes.

Transcriptomic Analysis Revealed the Regulatory Mechanisms of Oocyte Maturation and Hydration in Orange-Spotted Grouper (Epinephelus coioides).

Oocyte maturation and hydration are regulated by a complex interplay of various hormones and local factors. We have investigated the morphological changes of follicles and serum steroid levels during the HCG (human choionic gonadotophin)-induced oocyte maturation in the orange-spotted grouper. For the first time, a large-scale transcriptomic analysis of follicles during the maturation has been conducted in a fish species which produce pelagic oocytes. Eight cDNA libraries of follicle samples, from full-grown immature follicles to mature follicles, were constructed. A total of 402,530,284 high-quality clean reads were obtained after filtering, 79.66% of which perfectly mapped to the orange-spotted grouper genome. Real-time PCR results of 12 representative genes related to oocyte maturation and hydration verified the reliability of the RNA-seq data. A large number of genes related to oocyte maturation and hydration were identified in the transcriptome dataset. And the transcriptomic analysis revealed the dynamic changes of the steroid synthesis pathway and the pathway of hydration during oocyte maturation. The present study will facilitate future study on the oocyte maturation and hydration in the orange-spotted grouper and other marine pelagic egg spawner.

GHRH, PRP-PACAP and GHRHR Target Sequencing via an Ion Torrent Personal Genome Machine Reveals an Association with Growth in Orange-Spotted Grouper (Epinephelus coioides).

Growth hormone-releasing hormone (GHRH) and the receptor, GHRHR, constitute important components of the hypothalamus-pituitary growth axis and act on the downstream growth hormone (GH). PACAP-related peptide/pituitary adenylate cyclase activating polypeptide (PRP-PACAP) is a paralog of GHRH. These genes all play key roles in development and growth patterns. To improve the quality of cultured fish strains, natural genetic variation must be examined and understood. A mixed linear model has been widely used in association mapping, taking the population structures and pairwise kinship patterns into consideration. In this study, a mass cross population of orange-spotted grouper (Epinephelus coioides) was examined. These candidate genes were found to harbor low nucleotide diversity (θw from 0.00154 to 0.00388) and linkage disequilibrium levels (delay of 50% within 2 kbp). Association mapping was employed, and two single-nucleotide polymorphisms (KR269823.1:g.475A>C and KR269823.1:g.2143T>C) were found to be associated with growth (false discovery rate Q < 0.05), explaining 9.0%-17.0% of the phenotypic variance. The association of KR269823.1:g.2143T>C was also found via haplotype-based association (p < 0.05). The identified associations offer new insights into gene functions, and the associated single-nucleotide polymorphisms (SNPs) may be used for breeding purposes.

Expression profiles of gonadotropins and their receptors during 17α-methyltestosterone implantation-induced sex change in the orange-spotted grouper (Epinephelus coioides).

It is known that the hypothalamic-pituitary-gonadal axis participates in the sex change of hermaphrodite teleosts, and gonadal steroid hormones mediate this physiological process. The secretion of gonadal steroids is directly regulated by signaling pathways involving gonadotropins (GtHs) and gonadotropin receptors (GtHRs) in teleosts. To gain insight into the involvement of GtH/GtHR systems in the sex change process, cDNAs encoding follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were firstly isolated from gonads of orange-spotted grouper (Epinephelus coioides), a protogynous hermaphrodite fish. Reverse transcription-PCR (RT-PCR) analysis demonstrated that the expression of the FSHR was confined to the brain, pituitary gland, ovary, and testis, while the LHR was expressed only in the brain, ovary, and testis. Furthermore, the expression profiles of GtH subunits (FSHß and LHß) and their receptors were analyzed in parallel with the serum levels of estradiol-17ß (E(2) ), testosterone (T), and 11-ketotestosterone (11-KT) during 17α-methyltestosterone (MT)-induced sex change. Quantitative real-time PCR determined that the abundances of FSHß and FSHR were significantly inhibited after MT treatment for 2 and 4 weeks, but subsequently returned to the control level after 6 weeks. In contrast, the mRNA levels of LHß and LHR were significantly elevated throughout the sex change process. During MT-induced sex change, serum concentrations of E(2) remained constant while T and 11-KT levels were significantly increased. Taken together, our results suggest that GtH/GtHR systems are involved in MT-induced sex change, and two signaling pathways may have distinct roles in modulating the variations of the corresponding steroid hormones in the orange-spotted grouper.

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides).

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235bp for gERα, 1967bp for gERß1 and 2158bp for gERß2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERß1 mainly expressed in immature ovaries and gERß2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERß1 increased gradually, and gERß2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.

Molecular identification of the Kiss2/Kiss1ra system and its potential function during 17alpha-methyltestosterone-induced sex reversal in the orange-spotted grouper, Epinephelus coioides.

The Kiss1/Kiss1r system is a component of the hypothalamus-pituitary-gonadal (HPG) axis, which plays a crucial role in regulating gonadotropins and gonadotropin-releasing hormone. The sex reversal process is a special reproductive phenomenon regulated by the HPG axis. To better understand the neuroendocrine mechanisms of sex reversal, cDNAs encoding kiss2 and kiss1ra have been cloned and functionally characterized from the orange-spotted grouper Epinephelus coioides, a protogynous hermaphroditic teleost. The core mature peptide (Kiss2-10) of grouper Kiss2 shared high similarity to other KISS orthologs. In phylogenetic analyses, the grouper Kiss was clustered with the teleost Kiss2 clade and termed grouper kiss2. The predicted amino acid sequence of grouper kiss1ra contained three putative glycosylation sites at its N-terminus, showing high similarity to that of other teleosts. Synthesized Kiss2-10 was able to functionally interact with Kiss1ra in cultured COS-7 cells to trigger downstream signaling. Both kiss2 and kiss1ra mRNAs were expressed in all tissues examined, with highest levels in the olfactory bulb and moderate levels in the hypothalamus among brain areas and highest levels in ovary among peripheral tissues. Intraperitoneal injection of Kiss2-10 significantly increased gnrh1 mRNA levels in hypothalamus and follicle-stimulating hormone beta (fshb) mRNA levels in the pituitary at 6 and 12 h postinjection. During the process of sex reversal induced by 17 alpha-methyltestosterone (MT), kiss2 and kiss1ra mRNA expression were significantly decreased in the first week, but kiss2 increased in the fourth week, in accordance with the expression pattern of gnrh1 mRNA in the grouper hypothalamus. This is the first description of the Kiss2/Kiss1ra system during MT-induced sex reversal in orange-spotted grouper.

The second prolactin receptor in Nile tilapia (Oreochromis niloticus): molecular characterization, tissue distribution and gene expression.

Prolactin (PRL) is one of the most versatile hormones found in the pituitary of vertebrates and exerts its actions through binding to a specific PRL receptor (PRLR). Here we describe the cloning and characterization of a second prolactin receptor (ntPRLR2), isolated from the ovary of Nile tilapia (Oreochromis niloticus). The newly identified PRLR cDNA was 2011 bp in length and encoded 529 amino acids. It shared 31.6% identity in nucleotide sequence and 29.2% in deduced amino acid sequence with the first PRLR identified in Nile tilapia (ntPRLR1). Both of these ntPRLRs resemble the long form mammalian PRLRs. The nominated ntPRLR2 was further confirmed as a real prolactin receptor based on its competence to transactivate the beta-casein and c-fos promoters in the transiently ntPRLR2-transfected HEK293 cells. The ntPRLR2 gene also found to encode a 864-bp short form transcript in the ovary, which was confirmed by Northern blot analysis. A tissue distribution study by real-time PCR revealed that the mRNA of both receptors (ntPRLR1 and ntPRLR2) was widely expressed in different tissues, with an extremely high abundance in the osmoregulatory organs, including the gills, intestine and kidney. ntPRLR1 mRNA was more abundant than ntPRLR2 in the testis, while the reverse expression pattern was found in the ovary. In the ovary, ntPRLR2 mRNA demonstrated a distinct gonadal development-dependent expression profile, with significantly higher levels at a sexual mature stage than at sexual recrudescent and sexual regressed stages. When challenged with estradiol, ntPRLR2 mRNA expression was up-regulated by E2, whereas E2 had no significant effect on ntPRLR1.

Molecular cloning and functional characterization of the first non-mammalian 26RFa/QRFP orthologue in Goldfish, Carassius auratus.

In this study, we used data mining approach to predict 26RFa/QRFP precursors from fish, amphibian, reptile and avian species and subsequently cloned a 26RFa/QRFP precursor cDNA from goldfish brain based on the predicted sequences information. The goldfish 26RFa/QRFP precursor cDNA encoded a propeptide of 168 amino acids (aa) with predicted signal peptide of 30 aa at N-terminal and putative mature peptides, including 26RFa (26 aa) and 7RFa (7 aa) located at the C-terminal. Multiple sequence alignment showed almost all of the 26RFa/QRFP mature peptides possessed KGGFXFRF-amide motifs (X=G, S, A or N) at their C-terminus, and the last three residues FRF were fully conserved across vertebrates, indicating that the evolutionary pressure has exerted to conserve several C-terminal amino acid residues among the known and predicted 26RFa/QRFP precursors. Real-time PCR revealed that 26RFa/QRFP gene was expressed abundantly in goldfish hypothalamus, optic tectum-thalamus and testis. The regulation of goldfish hypothalamic 26RFa/QRFP gene expression by negative energy balance and putative role of goldfish 26RFa/QRFP in the control of luteinizing hormone (LH) release were studied. Hypothalamic 26RFa/QRFP gene expression was pronouncedly increased at 4 days after food deprivation. Furthermore, intraperitoneal (IP) injection of synthesized goldfish 26RFa/QRFP at a dose of 1 microg/g bodyweight significantly increased serum LH levels at 1h. However, LH levels were not significantly changed by IP injection of goldfish 26RFa/QRFP at lower dosage or at other time points (3 and 6 h), or by incubation of goldfish primary cell cultures. These results suggested that goldfish 26RFa/QRFP shared some similar features with its mammalian counterparts and partly exerted the regulatory function in energy homeostasis and hypothalamic-pituitary-gonadal (HPG) axis as observed in mammalian species.