|Other Abstract||Chinese sturgeon (Acipenser sinensis Gray) is an endangered anadromous fish, and was listed as a Grade I protected animal in China. It mainly reproduces in Yangtze River. Its reproduction migratory route was blocked in 1981 by the Gezhouba Dam, which caused a drastic decline of the natural population. However, its molecular information is very limited at this time. In order to isolate specific functional genes involved in reproduction and endocrine regulation and to reveal their evolutionary mechanisms in Chinese sturgeon, we constructed its pituitary cDNA library. In teleost, pituitary plays significant roles in many physiological functions, such as growth and reproduction. In this study, the SMART cDNA plasmid libraries of the pituitaries from 4-years old male Chinese sturgeon and 24-years old female Chinese sturgeon were constructed respectively. 944 and 2025 ESTs were obtained respectively from the two pituitary plasmid libraries.
Among all the 944 EST clones of the 4-years old male Chinese sturgeon, 461 (48.83%) clones were identified as known genes and 483 (51.17%) as unknown genes. The 461 clones of known gene products represent transcripts of 132 genes. Functional categorization indicated that the most abundantly expressed functional gene, the proopiomelanocortin (POMC), accounted for almost 10.17% of the overall expression, indicating its important function in the pituitary. Interestingly, the expression patterns of 7 unknown genes were analyzed in various tissues. Several unknown ESTs, such as EG009334, EG009337, EG009338 and EG009340, were detected to be pituitary-specific, or pituitary and ovary-specific genes.
Clustering and assembly of the 2025 ESTs of the 24-years old female Chinese sturgeon revealed 1305 ESTs that match with some known genes and 720 ESTs that have no significant homology to any sequences. And, a total of 881 different genes were identified. The known genes were categorized into 10 groups according to the predicted and unknown functions. Additionally, 75 different genes, including 20 contigs and 55 singletons, were identified from the 720 unannotated ESTs. Moreover, we found 8 reproduction and endocrine-related genes, and the 8 genes accounted for 557 in the 2025 ESTs. Interestingly, we observed 2 subtypes of gonadotropin subunit common α (GTH-α) and 2 subtypes of proopiomelanocortin (POMC), and found that they were the most redundant expression genes. They accounted for 16.6% and 7.3% of all the sequenced ESTs, respectively. Furthermore, we analyzed their phylogenetic significance and suggested that AsGTH-α II might be an ancestral gene, and might play significant roles in reproduction regulation of ancestral fishes. Additionally, GnRH receptor was firstly identified from sturgeons of Acipenseriformes.
The nucleotide sequences of Chinese sturgeon growth hormone (AsGH) cDNA contains 954 nucleotides and encodes 214 amino acid residues (aa). High homology were observed by amino acid sequence alignment and identity comparison of the mature peptides of AsGH with that of other fishes. RT-PCR and Western blot analysis showed that a considerable high level of GH transcript has been detected in the 14 embryo stages. These findings suggest that the AsGH in eggs may play a key role in early development of Chinese sturgeon
The nucleotide sequences of Chinese sturgeon common α I (AsGTH-α I), common α II (AsGTH-α II), FSHβ (AsFSHβ) and LHβ (AsLHβ) subunit cDNAs are 659, 642, 1101 and 584 bp in length, and encode for mature peptides of 115, 121, 129 and 138 aa, respectively. Three polyclonal antibodies were prepared from the in vitro expressed AsGTH-α I, AsFSHβ and AsLHβ mature proteins, respectively. A western blot analysis showed that the expression of the three gonadotropin subunits (AsGTH-α I, AsFSHβ and AsLHβ) can be detected in the individuals with 17-years-old (sex matured). While in other individuals, only AsFSHβ expressed in the pituitary of 4-years-old sturgeon. Immunofluoresence localization were performed and indicated that in the pituitary of 24-years-old individual (sex matured), the AsFSHβ cells mainly distributed in the middle area of proximal pars distalis (PPD), while the AsLHβ cells distributed in the area of PPD, pars intermedia (PI) and the external periphery of pituitary near to the side of rostral pars distalis (RPD). The AsGTH-α I might be expressed in both AsFSHβ and AsLHβ cells. In the pituitary of 4-years-old individual, the AsFSHβ cells mainly distributed in the external periphery of pituitary near to the side of RPD and PPD. Compared with the 24-years-old sturgeon’s pituitary, AsFSHβ cells distributed more widely in the pituitary of 4-years-old sturgeon.
Our results provide the basis for future investigations on reproduction biology of Chinese sturgeon.|