Molecular characterization of three gonadotropin subunits and their expression patterns during ovarian maturation in Cynoglossus semilaevis

Abstract

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHβ, LHβ and CGα) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHβ, LHβ and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHβ transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHβ expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole.

Figure 1

Representative stained sections for each of the five stages of C. semilaevis ovarian development. (A) previtellogenesis, ovary in stage II, bar = 100 μm; (B) vitellogenesis, ovary in stage III, bar = 100 μm; Balck arrow denotes vitellogenic oocyte; (C) late vitellogenesis, ovary in stage IV, bar = 200 μm; and (D) maturation, ovary in stage V, bar = 200 μm; (E) After ovulation, ovary in stage VI, bar = 200 μm; Balck arrows indicate post-ovulatory follicles or atretic oocyte. VO: Vitellogenic oocyte; PF: Post-ovulatory follicles; and AO: Atretic oocyte.

Figure 1

Representative stained sections for each of the five stages of C. semilaevis ovarian development. (A) previtellogenesis, ovary in stage II, bar = 100 μm; (B) vitellogenesis, ovary in stage III, bar = 100 μm; Balck arrow denotes vitellogenic oocyte; (C) late vitellogenesis, ovary in stage IV, bar = 200 μm; and (D) maturation, ovary in stage V, bar = 200 μm; (E) After ovulation, ovary in stage VI, bar = 200 μm; Balck arrows indicate post-ovulatory follicles or atretic oocyte. VO: Vitellogenic oocyte; PF: Post-ovulatory follicles; and AO: Atretic oocyte.

Figure 2

Changes of the Gonadosomatic Index (GSI) values at different ovary stages in C. semilaevis (n = 4). Each bar represents the mean ± standard error (SE). The values with different letters differ significantly from each other (p < 0.05).

Figure 3

Nucleotide and deduced amino acid sequences of C. semilaevis gonadotropin subunits (FSHβ (A), LH β (B) and CGα (C)). Nucleotides (upper sequence) and amino acid (lower sequence) are numbered on the left. Amino acids that comprise the signal sequence are underlined. The start and stop codon are shown by boxes. The stop codon (TAA or TGA) is indicated by asterisks (*). The putative polyadenylation signal (AATAAA or ATTAAA) is underlined.

Figure 4

Alignment of amino acid sequences of C. semilaevis mature FSHβ (A), LHβ (B) and CGα (C) subunits with those of representative vertebrates. The cysteine residues are marked by gray shadow and numbered from the N terminus. The N-linked glycosylation sites are indicated by black shadow. Sole: Cynoglossus semilaevis, Senegalese: Solea senegalensis, Halibut: Hippoglossus hippoglossus, Flounder: Paralichthys olivaceus, Seabass: Dicentrarchus labrax, Grouper: Epinephelus coioides, Seabream: Pagrus major, Mtilapia: Oreochromis mossambicus, Killifish: Fundulus heteroclitus, Goldfish: Carassius auratus, Zebrafish: Danio rerio, Trout: Oncorhynchus mykiss, Eel: Anguilla anguilla, Sturgeon: Acipenser baerii, Catfish: Clarias gariepinus, Stickleback: Gasterosteus aculeatus, Ntilapia: Oreochromis niloticus, Human: Homo sapiens.

Figure 4

Alignment of amino acid sequences of C. semilaevis mature FSHβ (A), LHβ (B) and CGα (C) subunits with those of representative vertebrates. The cysteine residues are marked by gray shadow and numbered from the N terminus. The N-linked glycosylation sites are indicated by black shadow. Sole: Cynoglossus semilaevis, Senegalese: Solea senegalensis, Halibut: Hippoglossus hippoglossus, Flounder: Paralichthys olivaceus, Seabass: Dicentrarchus labrax, Grouper: Epinephelus coioides, Seabream: Pagrus major, Mtilapia: Oreochromis mossambicus, Killifish: Fundulus heteroclitus, Goldfish: Carassius auratus, Zebrafish: Danio rerio, Trout: Oncorhynchus mykiss, Eel: Anguilla anguilla, Sturgeon: Acipenser baerii, Catfish: Clarias gariepinus, Stickleback: Gasterosteus aculeatus, Ntilapia: Oreochromis niloticus, Human: Homo sapiens.

Figure 5

Phylogenetic tree based on the amino acid sequences of FSHβ (A), LHβ (B) and CGα (C) from vertebrate. The tongue sole FSHβ, LHβ and CGα are highlighted by boldface. Bootstrap values (%) are indicated by 1000 replicates.

Figure 5

Phylogenetic tree based on the amino acid sequences of FSHβ (A), LHβ (B) and CGα (C) from vertebrate. The tongue sole FSHβ, LHβ and CGα are highlighted by boldface. Bootstrap values (%) are indicated by 1000 replicates.

Figure 6

Relative mRNA expression levels in maturing female C. semilaevis tissues. The abundance of FSHβ (A), LHβ (B) and CGα (C) transcripts, respectively, was determined by qRT-PCR and normalized to 18S (n = 4). Each bar represents the mean ± SE. Tissues marked by the different letters differing significantly from each other (p < 0.05). B: Brain, P: Pituitary, G: Gill, H: Heart, HK: Head kidney, K: Kidney, L: Liver, S: Spleen, St: Stomach, I: Intestine, O: Ovary, M: Muscle.

Figure 6

Relative mRNA expression levels in maturing female C. semilaevis tissues. The abundance of FSHβ (A), LHβ (B) and CGα (C) transcripts, respectively, was determined by qRT-PCR and normalized to 18S (n = 4). Each bar represents the mean ± SE. Tissues marked by the different letters differing significantly from each other (p < 0.05). B: Brain, P: Pituitary, G: Gill, H: Heart, HK: Head kidney, K: Kidney, L: Liver, S: Spleen, St: Stomach, I: Intestine, O: Ovary, M: Muscle.

Figure 7

Relative mRNA expression levels of FSHβ, LHβ and CGα mRNA at different ovary stages in maturing C. semilaevis. Abundance of FSHβ (A), LHβ (B) and CGα (C) transcripts, respectively, was determined by quantitative real-time PCR and normalized to 18S (n = 4). Each bar represents the mean ± SE. Tissues marked by the different letters differ significantly from each other (p < 0.05). The superscript “'” denotes the letter of statistical significance difference in the pituitary at different ovary stages. And the superscript “''” denotes the letter of statistical significance difference in the ovary at different.

Figure 8

Concentration of plasma FSH (A) and LH (B) at different ovary stages in maturing C. semilaevis (n = 4). Each bar represents the mean ± SE. The different letters differ significantly from each other (p < 0.05).