Effects Of GDF9and BMP15during The Developmental Process Of Oocyte Of Gibel Carp (Carassius Auratus Gibelio)

Transforming growth factor–β (TGF–β) superfamily is the intracellular maximalsignal protein family, its members are kinds of structurally conserved growth factors,they have been shown to participate in many physiologic activities such as cellularproliferation and differentiation and play important roles in embryonic development.Growth differentiation factor9(GDF9) and bone morphogenetic protein15(BMP15)are two oocyte–derived factors that belong to the TGF–β superfamily. Although theyhave key regulative functions during follicles development in mammals and birds,potential ovarian roles of them remain unexplored in teleosts.In this study, the full–length genomic DNA sequences of GDF9and the full–lengthcDNA sequences of BMP15were obtained using rapid–amplification of cDNA ends(RACE) and long–distance PCR (LD–PCR). The full–length genomic DNA sequencesof GDF9is3979bp, coding428amino acid residues which contain a specific “RKKR”protease cleavage site of TGF–β superfamily. The full–length cDNA sequence ofBMP15is2217bp, coding385amino acid residues which contain a specific “RIRR”protease cleavage site. Sequence analysis showed that gibel carp GDF9and BMP15amino acid sequences were similar to zebrafish and other bony fishes. Spatio–temporalexpression patterns of gibel carp GDF9and BMP15were analyzed using real–timequantitative PCR. The results revealed that GDF9and BMP15mRNA were largelyexpressed in ovary and testis, GDF9was slightly expressed in muscle and liver, BMP15was slightly expressed in muscle, liver, pituitary and brain, and they were not obviouslyexpressed in other tissues. GDF9and BMP15are mainly present at stage I follicles andtheir expression levels are gradually reduced with the development of follicles. Thesame result was obtained in immunohistochemistry localization of GDF9and BMP15proteins. Within the follicles, GDF9and BMP15mRNA were mostly expressed inoocytes but barely expressed in follicle layer cells. Treated with human chorionic gonadotropin (hCG) at isolated follicles of gibel carp in vitro showed that hCG had theeffect on promoting follicle maturation, and the stimulatory effect of gonadotropin onGDF9and BMP15mRNA expression exhibited strong time–and dose–dependence.When treated with5ng/mL and10ng/mL hCG with10h, the expression level of GDF9and BMP15were significantly increased, respectively. However, with the cultivation,the expression level of GDF9and BMP15mRNA had a decreasing trend. The results ofthis study indicated that GDF9and BMP15genes played a key role during developmentof follicles in gibel carp, especially in the early stage follicles.Base the preliminary researches on the structure, expression and function of GDF9and BMP15, this study researched the structure and expression pattern of six type Ⅰand Ⅱ receptors of GDF9and BMP15during the process of TGF–β superfamily signaltransduction pathway. The results revealed that the length of GDF9type Ⅰ receptorsgenes TGF–βR1a and TGF–βR1b were503bp and522bp, the length of BMP15typeⅠ receptors genes BMPR1Ba and BMPR1Bb were both895bp, which containedconservative specific “GS” domain of type Ⅰ receptors genes,“SGSGSG”, and thelength of GDF9and BMP15type Ⅱ receptors genes BMPR2a and BMPR2b were1085bp and564bp. Sequence analysis showed that six gibel carp receptors genes amino acidsequences were all similar to zebrafish. Semiquantitative RT–PCR analysis showed thatTGF–βR1b, BMPR1Bb and BMPR2a genes were mostly expressed in brain, butTGF–βR1a, BMPR1Ba and BMPR2b genes were mostly expressed in liver, also the sixgenes were slightly expressed in other tissues. Real–time quantitative PCR analysisshowed that the expression level of TGF–βR1a, BMPR1Ba and BMPR1Bb genes werefirst decreased and then increased during development of follicles, the expression levelof TGF–βR1b was significantly increased at the late stage of follicles development, butthe expression level of BMPR2a and BMPR2b were gradually decreased with thedevelopment of follicles. Within the follicle, TGF–βR1b, BMPR1Bb, BMPR2a andBMPR2b mRNA were mostly expressed in follicle layer cells, but TGF–βR1a andBMPR1Ba mRNA were expressed in both oocytes and follicle layer cells. These resultsabove not only confirmed the functions of GDF9and BMP15in the early stage follicles,but also indicated that GDF9and BMP15might make a regulative effect during thedevelopment of follicles using a potential paracrine signal pathway. This study providedsome information for clarifying the functions and signal transduction pathway of GDF9and BMP15.