Research note: Unveiling the impact of ovotransferrin on chicken primordial germ cells biological processes.

Xin Liu: Validation, Investigation, Formal analysis, Writing – original draft. Yixiu Peng: Validation, Investigation, Formal analysis, Writing – original draft. Jun Wu: Validation, Investigation, Formal analysis. Hongwu Qian: Validation, Investigation, Formal analysis. Xiaoqian Lv: Validation, Investigation, Formal analysis. Fan Li: Validation, Investigation, Formal analysis. Kai Jin: Data curation, Resources, Methodology. Yingjie Niu: Data curation, Resources, Methodology. Jiuzhou Song: Data curation, Resources, Software. Wei Han: Data curation, Resources, Software. Guohong Chen: Methodology, Supervision, Project administration, Writing – review & editing, Funding acquisition. Bichun Li: Methodology, Supervision, Project administration, Writing – review & editing, Funding acquisition. Qisheng Zuo: Methodology, Supervision, Project administration, Writing – review & editing, Funding acquisition.

Most cell cultures require the addition of serum to the culture medium to maintain growth and proliferation. Previous studies have shown that BHK and HeLa cells cannot survive in serum-free culture media, but can proliferate when 25 types of hormones are added, indicating that hormones can substitute for the functions of serum. Chicken serum contains estradiol and ovotransferrin ( Hayashi and Sato, 1976 ). In this study, PGCs do not proliferate in the absence of chicken serum ( Fig 1 A). Subsequently, a serum-free group was used as the control, with the addition of ovotransferrin and estradiol. The results revealed that ovotransferrin at 0.5 mg/mL significantly promoted PGC proliferation ( Fig 1 B), suggesting a functional role in maintaining cell renewal and viability. Fig. 1 Effects of serum and ovotransferrin on the proliferation of PGCs. (A). Morphological observation of PGCs after 72 h of culture in the presence or absence of chicken serum. (B) Morphological observation of PGCs cultured in serum-free chicken medium with different concentrations of estradiol and ovotransferrin. Scale bar = 50 μm. (C–D) PCNA immunofluorescence staining evaluation of PGCs proliferation activity and statistical chart. Scale bar = 50 μm. Data are expressed as the mean ± SD of 3 independent experiments. Values within a column followed by different superscript letters differ significantly ( P < 0.05 ). Fig 1 Effects of serum and ovotransferrin on the proliferation of PGCs. (A). Morphological observation of PGCs after 72 h of culture in the presence or absence of chicken serum. (B) Morphological observation of PGCs cultured in serum-free chicken medium with different concentrations of estradiol and ovotransferrin. Scale bar = 50 μm. (C–D) PCNA immunofluorescence staining evaluation of PGCs proliferation activity and statistical chart. Scale bar = 50 μm. Data are expressed as the mean ± SD of 3 independent experiments. Values within a column followed by different superscript letters differ significantly ( P < 0.05 ). Proliferating Cell Nuclear Antigen (PCNA) serves as an indicator of cell proliferation and is involved in many crucial cellular processes, including cell cycle regulation and apoptosis. The binding of the PCNA-Cyclins-CDKs protein complex during mammalian cell division can lead to G1/S phase cell cycle arrest( Sheng, et al., 2019 ). Previous studies have shown that ovotransferrin treatment of mouse osteoblasts, cell cycle control was exerted, with an increase in S phase cells and a decrease in G0/G1 phase cells, indicating active DNA synthesis and cell proliferation ( Shang and Wu, 2018 ). We found that 0.5 mg/mL ovotransferrin increased the PCNA positivity rate ( Fig 1 CD) and elevated the S-phase proportion in the cell cycle ( Fig 2 A), both of which indicate enhanced cell proliferation. These results align with previous studies showing that ovotransferrin regulates the cell cycle and promotes PGCs proliferation. Fig. 2 Effect of ovotransferrin on the cell cycle, cell adhesion, redox ability, and apoptosis of PGCs. (A). Flow cytometry analysis of the cell cycle distribution of PGCs cultured with different concentrations of ovotransferrin. (B). qRT-PCR analysis of cell adhesion-related genes (ZO-1, Occludin, JAM, and Claudin-1). (C). The effect of different concentrations of ovotransferrin on ROS content in PGCs. (D-F). Total GSH, GSH, and GSSG content in PGCs cultured with different concentrations of ovotransferrin for 72 h. (G-H). Changes in MDA and SOD levels with different concentrations of ovotransferrin. (I). Flow cytometry analysis of apoptosis rates in PGCs cultured with different concentrations of ovotransferrin. Fig 2 Effect of ovotransferrin on the cell cycle, cell adhesion, redox ability, and apoptosis of PGCs. (A). Flow cytometry analysis of the cell cycle distribution of PGCs cultured with different concentrations of ovotransferrin. (B). qRT-PCR analysis of cell adhesion-related genes (ZO-1, Occludin, JAM, and Claudin-1). (C). The effect of different concentrations of ovotransferrin on ROS content in PGCs. (D-F). Total GSH, GSH, and GSSG content in PGCs cultured with different concentrations of ovotransferrin for 72 h. (G-H). Changes in MDA and SOD levels with different concentrations of ovotransferrin. (I). Flow cytometry analysis of apoptosis rates in PGCs cultured with different concentrations of ovotransferrin. Ovotransferrin can significantly reduce the adhesion of Chlamydophila psittaci. Oral administration of bovine lactoferrin, the mRNA expression of Claudin-1, Occludin , and ZO-1 in intestinal epithelial cells of weaned piglets was significantly increased ( Sienkiewicz, et al., 2022 ). In order to investigate the effect of different concentrations of ovotransferrin on PGCs cell adhesion, qRT- PCR was used to detect the expression of cell adhesion related genes. As shown in the Fig. 2 B, compared with the group lacking chicken serum, the mRNA expression of ZO-1, Occludin , and JAM in the 0.5mg/ml ovotransferrin group was significantly increased ( P < 0.05 ), but did not affect the expression of Claudin-1 . Our results suggest that appropriate concentrations of ovotransferrin can reduce the adhesion of PGCs, while both excessively high and low concentrations are detrimental to PGC development. The above results indicate that the lack of serum can decrease the adhesion ability of PGCs, while the 0.5mg/mL ovotransferrin can upregulate the adhesion ability. Ovotransferrin can inhibit oxidative damage induced by endometriosis by increasing GSH levels and reducing ROS levels ( Ren et al., 2020 ). It can also reduce ROS levels by enhancing the free radical scavenging activity of cells. Overexpression of ovotransferrin confers increased tolerance to oxidative stress ( Ibrahim et al., 2013 ) and can induce intracellular oxidative responses in chicken macrophages and neutrophils ( Xie et al., 2002 ). To investigate the effects of serum deficiency and varying concentrations of ovotransferrin on the redox status of PGCs, we measured several oxidative stress indicators. As shown in Fig. 2 C-H, after 72 h of culture with different concentrations of ovotransferrin, no significant change was observed in total GSH content, but the levels of ROS, GSSG, and MDA initially decreased and then increased, whereas SOD activity and GSH levels showed the opposite trend, first increasing and then declining. These findings suggest that an optimal concentration of ovotransferrin can enhance the antioxidant capacity of PGCs and reduce cellular oxidative damage. Ovotransferrin can regulate osteoclast apoptosis by modulating the BCL-2 family ( Shang and Wu, 2018 ). To investigate the effect of ovotransferrin concentration on PGCs apoptosis, we quantified the apoptosis rate using flow cytometry ( Fig. 2 I). The apoptosis rate of PGCs initially decreased and then increased with rising ovotransferrin concentrations. The 0.5 mg/mL ovotransferrin group showed a significantly lower apoptosis rate compared to the 0 mg/mL, 0.1 mg/mL, and 1 mg/mL groups ( P 0.05 ). These results indicate that ovotransferrin inhibits PGC apoptosis, with the strongest effect observed at 0.5 mg/mL. This study demonstrates that ovotransferrin at a concentration of 0.5 mg/mL significantly enhances the proliferation, adhesion, antioxidant capacity, and survival of PGCs under serum-free conditions. These findings provide new insights into the functional roles of ovotransferrin in regulating key biological processes in PGCs.

Primary cells were cultured for 70 d to successfully establish a cell line, and the cell line was frozen. The cell line was recovered before use. Cells were resuspended in FAcs medium, which consisted of calcium-free DMEM (Meilunbio-PWL037, Dalian, China) as basal medium with the addition of CaCl2, Ovalbumin (Sigma-C7902, Sigma-A5503, Shanghai, China), B-27 TM supplement, chicken serum, GlutaMax, 2-Mercaptoethanol, MEM NEAA, Sodium Pyruvate (Gibco-17504044, Gibco-16110082, Gibco-35050061, Gibco-21985023, Gibco-11140050, Gibco-11360070, Shanghai, China), FGF2, Sodium Heparin, Human Activin A (MCE-HY- P70600 , MCE-HY-17567A, MCE-HY- P70311 , Shanghai, China). The cell culture conditions were according to the method of Liu et al ( Liu, et al., 2024 ). Ovotransferrin was purchased from Sigma (Sigma-C7786); dilute with culture medium; Estradiol (MCE-HY-B0141) dissolved in DMSO. PGCs were inoculated in 24-well plates, cells were collected and washed after 72 h of treatment with different concentrations of ovalbumin, 5 × 10⁵ cells were resuspended with 100 μL of 1 × binding buffer, incubated with Annexin V-FITC and PI for 15 min at room temperature and protected from light, 1 × binding buffer was added, mixed, and then assayed immediately on the machine. Please refer to the Annexin V-FITC apoptosis detection kit for specific operating steps (40302ES60, YEASEN, Shanghai, China). Collect the cells, add 4% paraformaldehyde for fixation, after fixation, use 0.5% Triton X-100 for permeabilization and 10% FBS for sealing, add PCNA antibody (1:200, AS039, ABclonal, Wuhan, China) and incubate overnight at 4°C, the next day, add goat anti-rabbit IgG (A0264, ABclonal, Wuhan, China), and incubate for 2 hours at 37°C, protected from light. Subsequently, the slides were incubated with DAPI for 15 min, and were sealed with cedar oleoresin. Finally, the samples were examined using a fluorescence microscope. A cell pellet was thoroughly mixed with 1 mL of pre-cooled 70% ethanol gently to allow fixation at 4°C for overnight. Then the cells were settled through centrifugation at 1,000 × g for 5 min, and then resuspended with 1 mL of pre-cooled PBS. Then the cells were settled through centrifugation at 1,000 × g for 5 min. 10 μL of a propidium iodide stock solution and 10 μL of a RNase A solution were added to 0.5 mL of a staining buffer, and thorough mixing was conducted to prepare a propidium iodide staining solution for later use. 0.5 mL of the prepared propidium iodide staining solution was added to each cell sample, and thorough mixing was conducted gently to resuspend cells. The cells were incubated at 37°C in the dark for 30 min and then tested at an excitation wavelength of 488 nm. The DNA content analysis and the light scattering analysis were conducted for the cells by Flowjo analysis software. Please refer to the Cell cycle and apoptosis analysis kit for specific operating steps (40301ES60, YEASEN, Shanghai, China). Total RNA was extracted with 1 mL of TRIzol reagent according to the manufacturer's instructions. RNA was reverse transcribed to cDNA and the PCR cycle conditions were set as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The relative mRNA expression levels were quantified using the 2 -ΔΔCT method. Cells were collected and inoculated into 96-well plates, standards were added and mixed with the samples, GSH assay working solution was added, incubated at room temperature for 5 minutes, then 0.5 mg/mL Nicotinic Acid Dinucleotide Phosphate (NADPH) solution was added and mixed well, and the absorbance value at 405 nm was measured immediately using a microplate reader. Refer to GSH and GSSG assay kit (S0053; Beyotime, Shanghai, China) for details. Cell samples were collected, mixed with reagent C of MDA assay kit (KGA7101-50, KeyGEN BioTECH, Nangjing, China), heated at 100°C for 40 min, quickly cooled in ice water, centrifuged at 4°C and supernatants were taken, and readings were analyzed by the microplate reader set at 532 nm. Cells were collected by centrifugation, DCFH-DA was added, and incubated at 37°C for 2 h. Cells were then washed with serum-free cell culture medium to remove the DCFH-DA that did not enter the cells, followed by flow cytometry. Reactive Oxygen Species Assay Kit (50101ES01; Yeasen, Shanghai, China) was used for the assay according to the manufacturer's instructions. Centrifugation was conducted at 1,400 rpm for 6 min, supernatant was discarded, and a resulting PGCs pellet was retained. 0.3 mL to 0.5 mL of a buffer, and ultrasonic disruption was conducted with a power of 300 W in an ice water bath. During the ultrasonic disruption, an ultrasound was applied 4 times at an interval of 3 s to 5 s, and the ultrasound lasted for 30 s each time. Then an enzyme working solution and an enzyme dilution were added, and the assay was conducted with a microplate reader. Specific steps refer to Superoxide Dismutase (SOD) assay kit (A001-3-2, Nanjing Jiancheng Bioengineering Institute, China) Data are presented as mean ± standard error, and all experiments were repeated at least three times. After sorting the data using EXCEL, significance analysis was performed, and diagrams were generated using SPSS 19.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Significant differences between groups were analyzed using a one-sample t-test and one-way ANOVA. Statistical significance was set at P <0.05 .

Most cell cultures require serum to maintain cell growth. In the chicken PGCs culture system, the requirement for chicken serum cannot be replaced by fetal bovine serum ( FBS ). Chicken serum contains various avian-specific cytokines and factors, with higher levels of insulin, progesterone, estradiol, and estriol compared to FBS ( Whyte, et al., 2015 ). However, due to the inconsistent chemical composition of serum and the high risk of contamination, there are issues with reproducibility and studying the effects of specific components. To address these limitations, serum-free media ( SFM ) have emerged as a more consistent and defined alternative. Transferrin, a key component of many SFMs, supports cell survival and growth by mediating iron transport and contributing to antioxidant defense. Ovotransferrin, as the only soluble glycoprotein in the avian transferrin family, is abundantly present in plasma and egg white, and can be obtained without the need for animal slaughter. Ovotransferrin playes a central role in development of chicken embryonic cartilage and hypertrophic chondrocytes demonstrating significant growth-stimulating effects on various cell types under serum-free conditions. Ovotransferrin can clear superoxide radicals and regulate immunity, induce apoptosis of matured osteoclasts, accompanied by increased expression of Bim and Bad, but decreased expression of BCL-2. Although studies have been conducted on other cell types, the effect of ovotransferrin on chicken PGCs has not been investigated. In this study, the focus was on investigating its effects on cell proliferation, adhesion, oxidative stress response, and apoptosis. By elucidating its cellular effects, new insights have been provided for the molecular regulation of PGCs.

None of the authors involved in this research have any conflicts of interest to report.