Molecular Characterization Of Follicle-stimulating Hormone β Subunit (FSHβ) And Its Receptor (FSHR) In Chinese Alligator And Their Spatial-temporal Expression During The Female Reproductive Cycle

The Chinese alligator(Alligator sinensis) is an endangered species and indigenous to China. Though the issues with artificial incubation and breeding of the Chinese alligators have been successfully resolved, little is known about the regulation of its female reproductive cycle, oogenesis and the related mechanism which affects reproductivity.Reproduction in vertebrates is mainly under endocrine control of hypothalamus–pituitary–gonad axis. The follicle-stimulating hormone(FSH), a glycoprotein hormone, is the key reproductive hormones involved in gonadal development. FSH belongs to the glycoprotein hormone family along with thyroid-stimulating hormone(TSH), luteinizing hormone(LH) and chorionic gonadotropin(CG). They are composed of two different subunits designated as α and β. The α subunit is common among these hormones within a species and is structurally conserved among different species, whereas the β subunit is specific to each hormone and confers biological specificity. FSH is the central hormone regulating vertebrate reproduction, to promote the growth and development of gonads, to control gametogenesis and regulate gonadal endocrine functions. FSH exerts its biological activities through interaction with specific receptors(FSHRs).In the present study, The characterization of FSHβ and FSHR in the Chinese alligator was studied by RT-PCR and RACE, and its tissue distribution and mRNA expression changes during the female reproductive cycle were investigated by qRT-PCR. The results are as follows:The complete FSHβ cDNA consists of 843-bp nucleotides, including 120-bp nucleotides of the 5’-untranslated region(UTR), 396-bp of the open reading frame, and3’-UTR of 327-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHβwith a signal peptide of 18 amino acids followed by a mature protein of 113 amino acids.Its deduced amino acid sequence shares high identities with American alligator(100%) andbirds(89-92%). Phylogenetic tree analysis of the FSHβ amino acid sequence indicated that alligators cluster into the bird branch. Tissue distribution analyses indicated that FSHβmRNA is expressed in ovary, intestine and liver with the highest level in the ovary, while not in stomach, pancreas, heart, thymus and thyroid. Expression of FSHβ in ovary increases in May(breeding prophase) and peaks in July(breeding period), it is maintained at high levels through September, then decreases significantly in November(post-reproductive period) and remains relatively low from January to March(hibernating period).FSHR cDNA consists of 2643-bp nucleotides, including 78-bp nucleotides of the5’-untranslated region(UTR), 2088-bp of the open reading frame, and 3’-UTR of 477-bp nucleotides. It encodes a 695-amino acid precursor molecule of FSHR with a signal peptide of 17 amino acids followed by a mature protein of 678 amino acids. The mature protein of Chinese alligator FSHR displays typical features of the glycoprotein hormone receptor family, but also contains some remarkable differences when compared with other vertebrate FSHRs. The deduced amino acid sequence of the Chinese alligator FSHR shares identity of 85% with chinese softshell turtle, 84-87% with birds, 77-78% with mammals,67-73% with amphibians and 51-58% with fishes. Phylogenetic tree analysis of the FSHR amino acid sequence indicated that alligators cluster into the bird branch. Tissue expression analysis showed that Chinese alligator FSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid.Expression of FSHR in the ovary is high in May(breeding prophase) and peaks in July during the breeding period, where it is maintained at high levels through September(breeding anaphase). Expression decreases significantly in November(hibernating period) and then remains relatively low from January to March(hibernating period).This study expands our knowledge of FSHβ and FSHR phylogenetic evolution. These temporal changes in FSHβ and FSHR expression indicate that they may play an important role in promoting ovarian development during the reproductive cycle. In addition,knowledge of the structure of FSHβ and FSHR obtained from this study may aid further studies related to structure–function relationships. Our studies found that the hormone-binding domain of FSHR have high similarity in structure with those of mammalian and bird FSHRs, based on this information, it may be possible to increaseFSHβ levels in Chinese alligator by using mammalian and bird FSHβ, which could stimulate the ovary to produce more estrogen and improve the reproductivity. This result provide basic data for further study on the mechanisms that regulate Chinese alligator reproduction, which could be useful for maximizing the success of artificial breeding.