| The giant panda(Ailuropoda melanoleuca)is a rare and endangered animal in China.The low reproductive rate restricts the population and conservation of giant pandas.The low reproductive activity of panda may be related to the low levels of gonadotropins in vivo,such as follicle-stimulating hormone(FSH)and luteinizing hormone(LH).Although the xenogeneic exogenous gonadotropins have been used to induce empathema and ovulation of giant panda for many years in China,the repeated use of heterogenous hormones may induce the body to produce adverse reactions and influence its long-term effects.To solve this problem,this study aimed to prepare the recombinant FSH and LH proteins of giant panda,and examine their biological activities by using a luciferase reporter system.The main results of this study are summarized as follows:1.The coding regions of giant panda FSHβ,LHβ and GTHα genes were synthesized by gene synthesis technology and used to construct the eukaryotic expression plasmids(pc DNA3.1-FSHβ-T2A-GTHα and pc DNA3.1-LHβ-T2A-GTHα).Then,these expression plasmids were used to transfect the Chinese hamster ovary cell(CHO)for preparation of the recombinant giant panda FSH and LH proteins.Using Western blot,we demonstrated that the three subunits of panda gonadotropins are expressed in CHO cells and media.2.The coding regions of mouse FSH receptor(m FSHR)and LH receptor(m LHR)were amplified from ovary by RT-PCR and cloned into the pc DNA3.1(+)expression vector.Using p GL3-CRE-luciferase reporter system,we found that both recombinant human FSH and LH could activate mouse FSHR and LHR,respectively,and their activation can stimulate the intracellular c AMP-PKA signaling pathway.These findings indicate that CHO cells expressing FSHR/LHR can be used to monitor the biological activity of recombinant panda gonadotropins in vitro.3.Using CHO cells expressing mouse FSHR/LHR,we further examined whether the conditioned medium containing recombinant panda FSH and LH can specifically active mouse FSHR or LHR.The results showed that recombinant panda FSH and LH can activate mouse FSHR and LHR respectively,indicating that panda FSH and LH expressed in CHO cells can not only be secreted into the culture medium,but also be assembled into heterodimers and glycosylated properly,which display biological activity identical to the endogenous FSH and LH.In summary,this study focused on the preparation of recombinant panda FSH and LH protein in CHO cells.Our data,for the first time,clearly indicate that the recombinant bioactive panda FSH and LH have been obtained.The results from this study will not only establish a basis to prepare the recombinant panda FSH and LH in a large scale,but also provide a feasibility to induction of ovulation by using panda gonadotropins,thus being valuable for the resolution of the reproductive barriers and improvement of reproduction rate and population size of giant pandas in the future. |
