Major drugs and reagents
FSH-Fc fusion protein (code: X002; molecular weight = ~90 kDa) is a new type of protein produced by fusing the Fc fragment of the human IgG4 subtype with α and β subunits of FSH (Figure 1). It was commissioned by the United Power Bioengineering and Biotechnology (Production Batch No. X0020220171205; Beijing, China) and developed by Beijing Qikang Xingye Biopharma Technology Co., Ltd. (Beijing, China). Gonal-f was purchased from Merck Serono (specification: 5.5 μg, 75 IU; Darmstadt, Germany), pregnant mare serum gonadotropin (PMSG) was purchased from Beijing Solarbio Science and Technology (Product No. P9970; Beijing, China), and human chorionic gonadotrophin (hCG) was purchased from ProSpec-Tany TechnoGene Ltd. (Product No. hor-250; Rehovot, Israel).
Major equipment and instruments
The major equipment and instruments used in this study included the Roche Cobas®4000 automatic analyser (Model No. Cobas®4000; Roche Diagnostics, Shanghai, China), the Stereo Microscope (Model No. SMZ800N; Nikon Imaging Instrument Sales, Beijing, China), an ordinary scale (Model No. YP2002; Shanghai YuePing Scientific Instrument (Suzhou) Manufacturing, Jiangsu Province, China), and a real-time fluorescence-based quantitative PCR system (Model No. ABI 7500Fast; Thermo Fisher Scientific, Shanghai, China).
Experimental animals
The animals used in this study included 26-day-old, specific-pathogen free (SPF) grade female Sprague Dawley (SD) rats, 6–8-week-old female SD rats (SPF grade), and 21–24-day-old female Kun Ming (KM) mice (SPF grade) purchased from Beijing HFK Bioscience Co., Ltd (Experimental Animal Production License No. SCXK (Beijing) 2014-0004; Beijing, China). The SD rats and KM mice were housed in SPF-grade rooms under a constant temperature of 20–26 °C, constant humidity of 40–70%, and alternating 12:12 h light:dark cycle. Animals had free access to food and water. All animal experiments were approved by the Committee of Animal Experiments and Experimental Animal Welfare of Beijing Qikang Xingye Biopharma Technology Co., Ltd. (Grant No.: IACUC2018-0002), and in strict accordance with the 3R principle (Reduction, Replacement, Refinement).
Detection of germinal vesicle breakdown (GVBD) in mouse oocytes
The 21–24-day-old female KM mice were subcutaneously injected with PMSG (5 IU/animal), followed by euthanisation with CO2 at 46 h after injection to collect the ovaries, and isolate and process the cumulus-oocyte complexes (COCs) to eventually obtain the naked oocytes. The oocytes (n = 80–100 per group) were added to a culture medium containing Gonal-f or X002 at concentrations of 0.3, 3.0, 30.0, or 300.0 nmol/L [6] for 4 h at 37 ℃, after which the GVBD rates of the oocytes were compared between the same concentrations of Gonal-f and X002 [3]. The GVBD rate was counted according to the following equation: GVBD rate (%) = (the number of oocytes in GVBD phase + the number of oocytes in first polar body phase) / (total number of oocytes − number of dead and degenerated oocytes) × 100%.
Analysis of mouse COC expansion
The 21–24-day-old female KM mice were subcutaneously injected with PMSG (5 IU/animal) and euthanised with CO2 at 46 h after injection to collect the ovaries and isolate the COCs. The COCs (n = 80–100 per group) were then incubated with culture medium containing Gonal-f or X002 at concentrations of 0.3, 3.0, 30.0, or 300.0 nmol/L[6] for 14 h at 37℃, after which the expanded diameters of COCs were compared at the same concentrations of Gonal-f and X002. The total RNA of COCs in the different groups was extracted according to the manufacturer instructions of the RNAsimple Total RNA Kit (catalog number: DP419, TIANGEN Biotech (Beijing)Co.,Ltd), and reverse transcription was used to synthesize the cDNA according to the manufacturer’s instructions of the revertaid first strand cDNA synthesis kit (catalog number: K1622, Thermo Fisher Scientific Inc). The cDNA templates of different COC groups were used for RT-qPCR to measure the expression of key genes related to the maturation process of oocytes, namely amphiregulin (Areg), epiregulin (Ereg), betacellulin (Btc), pentraxin 3 (Ptx3), hyaluronan synthase 2 (Has2), prostaglandin-endoperoxide synthase 2 (Ptgs2), and tumour necrosis factor-inducible gene 6 (Tnfaip6), according to the instructions of AceQ qPCR SYBR Green Master Mix (catalog number: Q121-02, Vazyme Biotech Co.,Ltd) and using the primers of individual genes (Table 1) [6].
The individual gene expression levels were determined and normalised to the internal reference gene, β-actin. The relative expression level of each gene in the control group was set to 1. The 2(−ΔΔCt) method was used to calculate the relative expression level (fold-change) of the corresponding gene in the experimental group.
Pharmacokinetic tests
To evaluate the pharmacokinetics (PKs) of X002, female SD rats (n = 8 per group) were administered a subcutaneous injection of X002 (5, 15, or 45 μg/kg), and the peripheral blood was collected from the orbital venous plexus at 4, 8, 24, 48, 72, 120, 168, 240, 336, 504, and 672 h after X002 administration. The collected peripheral blood samples were coagulated and centrifuged at 2,000 ×g for 20 min at 4 °C before the rat serum was separated. The rat serum samples were analysed by enzyme-linked immunosorbent assay (FSH enzyme immumoassay test kit, minimum sensitivity: 2.5 mIU/ml, catalog number: BC-1029, BioCheck, Inc.) for X002 at different concentrations. The WinNonLin® v 6.4 (Certara Corp.) software was used to calculate the PK parameters.
Testing the rat ovarian weight gain
Following the guidelines of the Pharmacopoeia of the Peoples’ Republic of China (2015 edition), 26-day-old female SD rats were chosen to test acting function of X002. After randomly grouping the 26-day-old female SD rats (n = 10 per group) according to their body weight, the animals were subcutaneously injected with 3.0 pmol per (each animal * each injection) Gonal-f (7 injections, with an interval of 12 h between each injection), 1.6 pmol per (each animal * each injection) X002 (single injection), 3.0 pmol per (each animal * each injection) X002 (single injection), 6.0 pmol per (each animal * each injection) X002 (single injection), or 12.0 pmol per (each animal * each injection) X002 (single injection)[6]. 84 h after X002 treatment, all animals were also injected subcutaneously with 3.75 IU per (each animal * each injection) hCG (n = 10 rats per group). The rats were euthanised with CO2 at 96 h after the first administration of corresponding treatments to collect their venous blood samples, which were subjected to coagulation, followed by dissecting and weighing the ovaries. The estradiol (E2) and progesterone (P4) levels were quantified by rat E2 or P4 ELISA kit (Estradiol (rat) Elisa kit, catalog number: K3831, BioVision, Inc.; rat progesterone Elisa kit, catalog number: 80558, Crystal Chem Inc.), respectively.
Detection of superovulation in rats
After randomly grouping the 26-day-old female SD rats (n = 10 per group) according to their body weight, the animals were subcutaneously injected with 3.0 pmol per (each animal * each injection) Gonal-f (7 injections in total, with an interval of 12 h between two injections), 1.6 pmol per (each animal * each injection) X002 (single injection), 3.0 pmol per (each animal * each injection) X002 (single injection), 6.0 pmol per (each animal * each injection) X002 (single injection), or 12.0 pmol per (each animal * each injection) X002 (single injection)[6]. They were all subcutaneously injected with 20 IU per (each animal * each injection) hCG 84 h after the first administration of the corresponding treatment (n = 10 rats per group). The rats were euthanised with CO2 108 h after the first administration of corresponding treatments to isolate the rat fallopian tubes for the counting of oocyte numbers inside [6].
Statistical analysis
All data are presented as the mean ± standard error of the mean or mean ± standard deviation. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used for graph preparation, and the SPSS v22.0 software (IBM, Armonk, NY, USA) was used for one-way analysis of variance (one-way ANOVA) and two-way ANOVA. P < 0.05 was considered statistically significant.