G protein-coupled receptors (GPCRs) are a superfamily of cell-surface receptors which modulate a variety of cell functions through coupling to heterotrimeric G-proteins and regulating downstream effectors such as adenylyl cyclases, phospholipases, protein kinases and ion channels . FSHR belongs to the subfamily of GPCRs, which have great potential as therapeutic targets for abroad spectrum of diseases   . Follicle- stimulating hormone (FSH) was an important glycoprotein hormone to regulate reproduction in mammals, it acts through binding to follicle-stimulating hormone receptor (FSHR) on target cell surfaces  . The biological activities and half-life of FSH are associated with glycosylation, sialylation and sulfation  .
As the biotechnology has rapidly expanded in recent years, the majority of recombiant proteins have been produced by the genetic engineering technique using different host cell systems for the expression of heterologous genes such as Escherichia coli, Yeast, Insect/baculovirus, Mammalian cells, as well as Eukaryotic individuals, both of which have inherent advantages and drawbacks  . Besides, a variety of proteins analogues have been obtained via chemical synthesis   .
However, the bioactivity and physicochemical characteristics of the recombiant protein are not clearer in vitro or in vivo. At present, the mainly methods to detect the in vitro bioactivity for the recombinant follicle-stimulating hormone by the rat granulosa cell or the Sertoli cell aromatization bioassays   and follicle-stimulating hormone radioreceptor activity methods . In this study, we have adopted a methods of the pSNAPf-ADRβ2-FSHR  and cellular internalization  to determination and evaluation for the biological activities of recombinant follicle-stimulating hormone in vitro.
2. Materials and Methods
2.1. Reagents and Standards
The cloned human kidney 293T-FSHR cell strains (our laboratory), 96-well cell culture plates (NEST), SNAP-Surface™ 549 (NEB), DMEM (Hyclone), Fetal bovine serum (WISTEN), Bovine serum albumin(BSA, Sigma-Aldrich; USA), Puregon (Organon, Netherlands), Carbon dioxide incubator(Thermo), Fluorescence microscope (Bio-Rad).
2.2. The Cellular Internalization for rFSH in Vitro Activity Assay
A HEK-293T cell lines that had been stably transfected with the pSNAPf-ADRβ2-FSHR was used to detect fluorescent signal in response to the commercialization of recombinant follicle-stimulating hormone (rFSH) stimulation. Fluorescent signal were determined using imaging approaches, the total process of the cellular internalization as follows.
Cells were cultured in 96-well plates with 200 ul of DMEM supplemented with 10% fetal bovine serum (FBS). After 48-72 hours, replace the medium with 50 ul/well the labeling medium of 5 μM SNAP-Surface 549 (NEB) containing 0.5% bovine serum albumin (BSA) and incubate at 37˚C, 5% CO2 for 15 minutes. Wash the cells ten times with DMEM (Hyclone) containing 10% fetal bovine serum (FBS, WISTEN) and PBS (0.01 M, PH7.2), respectively. Adding 50 ul/well Puregon dilution (30 mIU/ml) and set the controls, respectively. Image the cells using the Fluorescence microscope (Bio-Rad) and continuously recording 0 min, 30 min, 60 min, 90 min, 120 min, 150 min.
The cellular internalization for rFSH in vitro activity assay, we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization. The control groups (Figure 1), after 90 min appeared a large number of fluorescence accumulated around the nucleus. However, the experimental groups (Figure 2), after 60 min have appeared the fluorescence accumulated around the nucleus. Suggested that the recombinant follicle-stimulating hormone promoted the fluorescent from the surface of the cells into the cytoplasm and accumulated around the nucleus. Meanwhile, the results indicated that this detection system was successed in determination and evaluation of the biological activities for rFSH in vitro.
Figure 1. The control goroups: 50 ul/well PBS, images acquisition from 0 min to 150 min.
Figure 2. The experimental groups: 50 ul/well pruegon dilution (30 mIU/ml), images acquisition from 0 min to 150 min.
The detection system based on the FSH receptor activation by FSH, FSHR induces intracellular cAMP synthesis, in turn, triggers downstream signal transduction  . To reduce background interference, used 0.5% bovine serum albumin (BSA) in the labeling medium and removed the SNAP-Surface 549 dye with DMEM containing 10% fetal bovine serum (FBS). Compared with the control groups, the commercialization of the rFSH have promoted the internalization of the fluorescence, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.
However, there are also many drawbacks such as low sensitivity, inconvenience in tracing images, and unsatisfied in recognising result. In the next study, we will further improve the detection system of bioactivity for rFSH and combined with cAMP-Screen cAMP Immunoassay System  .
This work was supported by these projects in China (31171731, 31460447, 20142- BDH80003, 2013-CXTD002, “555 talent project” of Jiangxi Province, Jiangxi Province Key Laboratory of Bioprocess Engineering and Co-Innovation Center for in vitro Diagnostic Reagents and Devices of Jiangxi Province).
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