Role of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 in Chicken Embryo Osteogenesis, Adipogenesis, Myogenesis, and Vitamin D3 Metabolism

A study was conducted to understand the effects of 25-hydroxyvitamin D3 (25OHD) and 1,25-dihydroxyvitamin D3 (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D3 metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses (n=8). Gene expression for key osteogenesis markers (RUNX2: runt-related transcription factor 2; BMP2: bone morphogenetic protein 2; COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatese), adipogenesis markers (PPAR-γ: peroxisome proliferator-activated receptor gamma; FASN: fatty acid synthase; and FABP4: fatty acid binding protein 4), myogenesis markers (MYOG: myogenin; MYOD1: myogenic differentiation 1; and MYF5: myogenic factor 5), and the enzyme responsible for vitamin D3 inactivation (CYP24A1: cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker, ALP, was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers (FAPB4 and FASN). In contrast, the expression of myogenesis markers (MYOD1 and MYF5) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker, SPP1, was increased by 25D-H. MYOD1 and MYF5 were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D3 metabolite retention was assessed by measuring CYP24A1 expression. At 1h, there were no differences in CYP24A1 expression. At 3h, all treatments upregulated CYP24A1 expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher CYP24A1 expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.


INTRODUCTION
Vitamin D 3 is essential for normal chicken embryo development (Sunde et al., 1978). It is well-established that Vitamin D 3 undergoes two biological conversions, first in the liver, to become 25-hydroxyvtamin D 3 (25OHD) hydroxylated by 25-hydroxylase (CYP2R1), and then mainly, in the kidney to become its biologically active form, 1,25-dihydroxyvitamin D 3 (1,25OHD), which is catalyzed by 1α-hydroxylase (CYP27B1; St-Arnaud, 2008;Christakos et al., 2010). Vitamin D 3 is primarily stored in the egg yolk in the form of 25OHD to be used by the developing chicken embryo during development (Ovesen et al., 2003;Vieira, 2007;Fatemi et al., 2020). In the poultry industry, 25OHD has become a commercial feed additive for poultry production due to its higher bioactivity than regular vitamin D 3 (Soares et al., 1995;Atencio et al., 2005a). Considerable research has demonstrated beneficial effects of 25OHD on chicken bone quality (Koreleski and Swiatkiewicz, 2005;Wideman et al., 2015;Chen et al., 2020) and muscle development (Michalczuk et al., 2010;Han et al., 2016).
In addition to utilizing vitamin D 3 metabolites in poultry feed, the influence of exogenous 25OHD during embryo development has also been studied. The administration of 25OHD can be achieved by in ovo injection of 25OHD directly to the yolk of chicken embryos at embryonic day 18 (ED 18), which has shown to reduce late embryo mortality (Fatemi et al., 2020). A less invasive alternative for increasing embryo 25OHD level has been accomplished by providing 25OHD in parent breeder diets. Studies have shown that this practice could significantly improve hatchability and embryo livability (Atencio et al., 2005b;Saunders-Blades and Korver, 2014). However, limited research has been conducted to evaluate the role of vitamin D 3 metabolites during early development of chicken embryos. Even though considerable cell culture studies have been performed to understand the vitamin D 3 signaling pathway in osteogenesis, adipogenesis, and myogenesis (Ding et al., 2012;Girgis et al., 2013;van Driel and van Leeuwen, 2014;Dix et al., 2018), the mono-cell culture model fails to account for interactions with alternate cell types or presence of extracellular factors such as fibroblast growth factors, insulin, and growth factors (Rosen and MacDougald, 2006). Osteoblasts, adipocytes, and myoblasts are all differentiated from embryonic stem cells (Kolf et al., 2007). The factors in favor of one cell fate may be at the cost of others (Kolf et al., 2007). As such, it is critical to study the role of vitamin D 3 in embryogenesis, while all different types of cells are present simultaneously.
Thus, the current study was conducted to understand the role of vitamin D 3 metabolites on the expression of key osteogenic, adipogenic, myogenic, and vitamin D 3 metabolism genes in the chicken embryos. Treatments were injected through the dorsal vein of chicken embryo at ED 3. Embryos were then sampled to examine expression of various genes related to osteogenesis, adipogenesis, and myogenesis. The current study provided an overview of how vitamin D 3 metabolites affect embryogenesis and the metabolism of vitamin D 3 in the embryo during early developmental stages. The results could also provide insight into the manipulation of nutrients during chicken embryo development and contribute to designing a strategy targeting optimized embryo nutrition.

Embryo Handling and Treatments
The study was approved by the Institutional Animal Care and Use Committee at the University of Georgia and conducted at the research facility of the Department of Poultry Science at the University of Georgia. Cobb 500 fertilized eggs were purchased from Cobb Hatchery (Cleveland, GA, United States). A total of 120 Cobb 500 fertilized eggs (8 eggs/treatment × 5 treatments × 3 time points) were used in the current study. They were incubated inside a bench incubator (GQF 1502, Savannah, GA, United States) at 37.5°C and around 45% relative humidity. The eggs were positioned horizontally without turning to ensure that the position of embryos was appropriate and consistent for injection. At 72 h of incubation, fertilized eggs were sprayed with 70% alcohol, and 1.5 ml albumen was carefully taken out from the sharp end of the eggs. The needle hole was sealed with glue immediately to avoid contamination. A mini drill was used for opening a window (around 1 cm 2 ) on the side of the eggs. Hundred microliter of Penicillin-Streptomycin (10,000 U/ml; Thermo Fisher Scientific, MA, United States) was applied to the eggs. Then the eggs were placed under a microscope (Olympus, PA, United States). PBS (C), PBS with 40 ng 1,25-dihydroxyvitamin D 3 (1,25D-L), 200 ng 1,25-dihydroxyvitamin D 3 (1,25D-H), 40 ng 25-hydroxyvitamin D 3 (25D-L), or 200 ng 25-hydroxyvitamin D 3 (25D-H; MilliporeSigma, MO, United States) were injected into the dorsal vein of embryos (Figure 1). Afterward, glass coverslips were used to seal the windows, and injected eggs were placed back to the incubator. The injected time was recorded for individual eggs. Whole embryos were harvested at 1, 3, and 6 h post-injection for gene expression analyses (eight embryos/  Bhat et al., 2014;Ji et al., 2015); key myogenesis marker genes (MYOG: myogenin; MYOD1: myogenic differentiation 1, and MYF5: myogenic factor 5; Wagatsuma and Sakuma, 2014); and a vitamin D catabolism gene (CYP24A1: cytochrome P450 family 24 subfamily A member 1; Christakos et al., 2010) were investigated (Table 1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a housekeeping gene. Samples were normalized and analyzed by the ΔΔCT method (Livak and Schmittgen, 2001).

Statistics
All experimental data were analyzed statistically by one-way ANOVA using SAS software Version 9.3 (SAS Institute, Cary, NC). Variability in the data was expressed as standard error mean (SEM). Differences between means were determined using Duncan's Multiple Range test. The level of significance was assessed at p ≤ 0.05.

RESULTS
The sequence for CYP27B1 (encoding 1α-hydroxylase) has not yet been identified in chickens (NCBI). 1α-hydroxylase is the critical enzyme that catalyzes 25OHD to 1,25OHD (Christakos et al., 2010). Thus, its expression to indicate the conversion of 25OHD to 1,25OHD could not be examined in the current study. Additionally, the level of 1,25OHD is difficult to measure in the embryo, because the sampling of embryo could not avoid breaking blood vessels, which could convolute analysis. However, the expression of CYP24A1 encoding 24-hydroxylase that is responsible for 1,25OHD inactivation to 1,25,24 OHD (van Driel and van Leeuwen, 2014) could be stimulated by 1,25OHD (Veldurthy et al., 2016). Thus, by investigating CYP24A1 expression level, we could observe the 1,25OHD residues status in the embryos. Since no differences were observed in BMP2, COL1A2, and BGLAP expression, these data were not shown in this manuscript. At 1 h post-injection, there was no difference in the vitamin D 3 metabolism marker (CYP24A1) expression (Figure 2A). Meanwhile, no significant differences were observed on osteogenesis (Figures 3A-C), adipogenesis (Figures 4A-C), and myogenesis marker gene expression (Figures 5A-C).
At 3 h post-injection, all treatments showed higher CYP24A1 expression than control (p = 0.002; Figure 2B), which indicated the injected 1,25OHD and 25OHD may have activated the catabolism of vitamin D 3 in the embryos. At the same time, early osteogenesis differentiation marker (ALP) was increased by high 1,25OHD and high 25OHD injection compared to the control (p = 0.0027; Figure 3E). However, no differences were found in RUNX2 ( Figure 3D) and SPP1 expression ( Figure 3F). With respect to genes involved in adipogenesis, high 25OHD injection induced the expression of FABP4 (p = 0.0351; Figure 4D) and FASN (p = 0.0339; Figure 4E) compared to the control group, although no differences were detected in the expression of PPAR-γ ( Figure 4F). Meanwhile, clear inhibitory effects of vitamin D injection on expression of genes regulating myogenesis were observed. 25OHD treatments significantly decreased MYOD1 expression at 3 h (p = 0.0224; Figure 5D), and 1,25OHD treatments, and high level 25OHD suppressed the expression of MYF5 (p < 0.0001; Figure 5E). No difference in MYOG expression was observed among treatments ( Figure 5F). At 6 h post-injection, CYP24A1 expression was significantly higher in 25D-H (p = 0.0003; Figure 2C), indicating a higher dosage of 25OHD possibly obtained a longer retention time. Meanwhile, 25D-H has higher SPP1 expression compared to the control (p = 0.0061; Figure 3I), but no difference was found in RUNX2 and ALP expression (Figures 3G,H). There were also no differences in adipogenesis gene expression (Figures 4G-I). However, 25OHD treatments down-regulated MYOD1 expression (p = 0.0008; Figure 5G), and both high 1,25OHD and 25OHD treatments suppressed MYF5 expression (p = 0.0210; Figure 5H). Similar to other time points, no difference in MYOG expression were identified at 6 h postinjection ( Figure 5I).

DISCUSSION
In the current study, 25OHD and 1,25OHD showed similar effects on embryogenesis, which indicated the embryo might have acquired the ability to convert 25OHD to 1,25OHD at ED 3. However, to our knowledge, the earliest time that the kidney of chicken embryos can produce 1,25OHD is reported at ED 9 (Bishop and Norman, 1975). Nevertheless, the mesonephros (intermediate kidney) begins to appear at about 55 h of incubation, and fully formed at ED 4 (Sturkie, 2012;Bolin and Burggren, 2013). It suggested that the intermediate kidney at this embryo stage could convert 25OHD to 1,25OHD (Kubota et al., 1981). Moreover, CYP27B1 (1α-hydroxylase) has reportedly been expressed in lymph nodes, skin, colon, pancreas, dendritic cells, brain, pulmonary alveolar macrophages, pathological parathyroid glands, prostate cells, and bones in various animal models (Panda et al., 2001;Zehnder et al., 2001;van Driel et al., 2006;Atkins et al., 2007;Adams and Hewison, 2012), suggesting that 25OHD may be converted by tissues outside of the kidney. A previous study has shown that 25OHD could inhibit the proliferation of myogenic cells in a similar manner to 1,25OHD, suggesting that the conversion of 25OHD to 1,25OHD may happen in myoblasts (Srikuea et al., 2012). However, further research needs to be conducted to validate the earliest time that chicken embryos can metabolize vitamin D 3 .
Both 1,25OHD and 25OHD are metabolized by CYP24A1 (24-hydroxylase), becoming 1,24,25OHD and 24,25OHD, respectively (Norman et al., 1980). 1,25OHD induces CYP24A1 synthesis via several pathways to form a negative feedback loop, avoiding excess 1,25OHD formation (Zierold et al., 1995;DeLuca, 2004). Thus, the expression of CYP24A1 could indicate the catabolism status of vitamin D 3 metabolites. Results indicated that the injection had not triggered the enhanced catabolism process at 1 h post-injection. However, at 3 h postinjection, all the treatments showed higher CYP24A1 expression compared to control group. At 6 h post-injection, only the higher level of 25OHD obtained the highest expression of CYP24A1. The catabolism of vitamin D 3 in the embryos was surprisingly fast, which may be attributed to the fact that catabolism of vitamin D 3 is tightly and rapidly regulated by 1,25OHD (Haussler et al., 2013). The current study showed the positive effects of vitamin D 3 metabolites on ALP (alkaline phosphatase) and SPP1 (osteopontin) expression at 3 and 6 h post-injection, respectively. ALP is an early osteogenesis marker and is essential for bone mineralization by enhancing the initial mineral crystal formation (Chatakun et al., 2014). SPP1 codes a highly phosphorylated sialoprotein with strong mineral-binding capacities in the extracellular matrix (Chabas, 2005). The role of vitamin D 3 metabolites on osteogenesis has been extensively investigated. However, cell culture studies on the influence of vitamin D 3 on osteogenesis have produced varied, and at times conflicting, results (van Driel and van Leeuwen, 2014). In agreement with our findings, 1,25OHD has been shown to stimulate on ALP expression in human osteoblasts (Matsumoto et al., 1991;Siggelkow et al., 1999;Chen et al., 2002;Woeckel et al., 2010). However, 1,25OHD is also reported to down-regulate ALP expression in mouse osteoblasts (Chen et al., 2012;Kim et al., 2016). Meanwhile, expression of Spp1 has been reported to increase in response to 1,25OHD in ROS 17/2.8 cell (rat; Staal et al., 1996). These inconsistent results may reflect different experimental conditions such as species, cell stage, treatment time, and dosages (van Driel and van Leeuwen, 2014). Besides the in vitro studies' contradictory results, additional vitamin D 3 or 25OHD injected to chicken embryos at ED 18 revealed a positive effect on bone quality of hatched chicks (Abbasi et al., 2017;Zamani et al., 2018). It is important to note that the cell culture system has limitations such as missing interactions of different types of cell and lacks consideration of other extracellular factors (van Driel and van Leeuwen, 2014). Indeed, it has been reported that factors, such as phosphate concentration, growth factors, and cytokines, may affect the function of 1,25OHD (van Driel and van Leeuwen, 2014).
The stimulatory effects of vitamin D 3 metabolites on adipogenesis at 3 h post-injection were observed in the current study. However, in previous studies, the effects of vitamin D 3 metabolites on adipocyte differentiation were inconsistent. Stimulatory effects were found in murine studies, but inhibitory effects were frequently shown in the humans (Dix et al., 2018). Other animal studies showed mixed results and are thoroughly reviewed in Dix et al. (2018). In chickens, an in vivo study showed that feeding additional 25OHD did not change the fat pad weight but did increase monounsaturated fatty acids and reduced the polyunsaturated fatty acids (Michalczuk et al., 2010). Even though the vitamin D 3 interactions with adipose tissue have been reported in various animal and human models (Ding et al., 2012), limited data are available on vitamin D 3 metabolites and chicken adipocytes. The research herein may contribute to our understanding of the fatty liver in old laying hens.
Both osteoblasts and adipocytes are differentiated from embryonic stem cells and mesenchymal stem cells (Kolf et al., 2007). A study has shown that PPAR-γ, the master regulator of adipogenesis (Kawai and Rosen, 2010), could alter mesenchymal stem cells (MSCs) fate by suppressing osteogenic transcription factors, such as homeobox protein DLX5, RUNX2, and Osterix (Kawai and Rosen, 2010), indicating a reciprocal relationship between osteogenic and adipogenic differentiation. Similar results were found in the previous study, where 1,25OHD increased adipogenic differentiation but inhibited osteoblastic cell proliferation and differentiation in rat bone marrow stromal cells (Atmani et al., 2003). However, in the current study, vitamin D 3 metabolites stimulated osteogenesis and adipogenesis simultaneously, evidenced by increased expression of FAPB4 and FASN (adipogenesis markers), and ALP (osteogenesis marker) at 3 h post-injection. This may reflect that the chicken embryo contains a number of extracellular factors, such as fibroblast growth factors, insulin, and growth factors, contributing to the complexity of vitamin D 3 metabolism on adipogenesis (Rosen and MacDougald, 2006). In the current study, the inhibitory effects of 1,25OHD on myogenic differentiation were shown as a decrease of MYOD1 and MYF5 expression at both 3 and 6 h post-injection. MYF5 and MYOD1 are families of Helix-Loop-Helix transcription factors that are expressed during myoblast proliferation and myotube differentiation (Bismuth and Relaix, 2010;Braun and Gautel, 2011). In agreement with the current study, VDR (vitamin D receptor) knockout mice had higher Myf5 and MyoG expression compared to the normal mice (Endo et al., 2003), which indicated the regulatory effects of vitamin D 3 on myoblast differentiation. On the contrary, it has been reported that 1,25OHD has a stimulatory effect on the proliferation and differentiation of embryonic chick myoblasts in culture, suggested by an increase in both cell density and fusion after 1,25OHD treatment ( Giuliani and Boland, 1984). Likewise, in mice, treatment of C2C12 (mouse myoblast) cells with 1,25OHD increased the MyoD1 and MyoG expression (Garcia et al., 2011). The current paper is contributing to the understanding of these species/cell specific data as it has its own set of unique variables and context. However, the specific pathways involved in such an effect in chicken embryo need to be investigated further. In summary, 25OHD and 1,25OHD administration to embryos elicited similar responses, suggesting that the embryo may be able to convert 25OHD to 1,25OHD; however, further research is necessary to determine the specific tissue location of the conversion. The catabolism of injected vitamin D 3 metabolites appeared to be remarkably fast based on the expression of a key vitamin D 3 catabolism-related gene (CYP24A1). The higher dosage of 25OHD showed a possibility of a longer retention time in the embryo. Additionally, both 1,25OHD and 25OHD increased the expression of osteogenesis and adipogenesis-related genes but inhibited myogenesis-related gene expressions during early embryo development in this study. However, the detailed pathways involved in these effects need further studies. Our findings provide an overview of the role of vitamin D 3 metabolites in early chicken embryogenesis and the potential basis of practical strategies of early nutrient supplementation in chicken embryos.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Georgia.

AUTHOR CONTRIBUTIONS
CC and DW: investigation. WK and CC: validation and data curation. CC: formal analysis, writing -original draft preparation, and visualization. WK, BM, and CC: writingreview and editing. WK: supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.