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Investigating the Effects of Fertilized Egg Yolk Extract on Myoblast Proliferation and Differentiation

Abstract

Muscle disorders like sarcopenia are characterized by compromised skeletal muscle mass and function. This is caused by the failure of satellite cells or myoblasts to fuse together to form myofibers, or decreases in their number and function. Therefore, any analysis of interventions for muscle wasting should observe their effects at the cellular level. There are a number of commercially available nutritional products that claim to increase muscle mass and health. One potential supplement is fertilized chicken egg yolk; it is rich in vitamins, minerals, fats, amino acids, and proteins which can provide a sustained supply of nutrition to the myoblasts. As reversing the effects of sarcopenia involve muscle tissue growth, and tissue growth is a prime component of regenerative/tissue engineering, one could evaluate the effectiveness of nutritional supplements using regenerative/tissue engineering tools. In the present study, we evaluated the effect of different concentrations of fertilized chicken egg yolk extract on the viability, morphology, and myogenic gene expression of C2C12 myoblasts, common tools in regenerative/tissue engineering and regenerative medicine. Egg yolk (fertilized and unfertilized) extract in media enhanced the proliferation and differentiation of myoblasts in a dose-ependent manner as observed by increased cell viability, number of nuclei, number of myofibers, and mRNA expression of muscle-specific genes such as MyoD and myogenin. There was no significant difference in cellular metabolism/proliferation between myoblasts exposed to fertilized and unfertilized egg yolk, but there were differences in cellular morphology and cell fusion between the two groups at specific concentrations.

Lay Summary

Muscle loss is frequently the result of aging or extreme sickness. While muscle loss and the effectiveness of any treatment is usually measured by physicians using visual observations of muscle tone or changes in weight, as this problem occurs at the cellular level, the effectiveness of any intervention should also be examined at the cellular level. Nutritional supplements are frequently used to combat muscle loss. One potential supplement is fertilized chicken egg yolk; it is rich in vitamins, minerals, fats, amino acids, and proteins which can provide a sustained supply of nutrition to muscle cells (myoblasts). In manuscript, we describe a study that tested the effectiveness of fertilized chicken egg as a way of combating muscle loss. We exposed myoblasts to different concentrations of fertilized egg yolk and looked at their effects on the behavior of the myoblasts. Exposure to egg yolk (fertilized and unfertilized) increased the number of cells and caused the cells to produce factors associated with muscle development in a dose-dependent manner. Exposure to fertilized egg yolk led to a more muscle-like shape in myoblasts (they fused together to form the multinucleated structures seen in muscle) compared to unfertilized egg yolk at specific concentrations.

Introduction

Dysfunctional muscle regeneration is characterized by reduced differentiation and fusion of satellite cells into myofibers or incomplete fusion of regenerated myofibers which lead to muscle disorders [1,2,3,4]. It is believed that the improvement of poor or dysfunctional muscle regeneration can be achieved by implementing new therapeutic strategies that are targeted at the cellular level. There are several aspects of skeletal muscle regeneration that individually and collectively contribute to regeneration of innervated and vascularized muscle tissue. These include the proliferation or differentiation of myogenic stem cells, regulation of myogenic regulatory molecules (such as myogenin and MyoD), and upregulation or downregulation of muscle-specific genes or aging [3,4,5,6,7,8].

Sarcopenia is a condition that causes age-related involuntary muscle loss which can begin at the age of 40. Once it begins, the decline of muscle strength and mass can occur in a linear manner and can lead to a loss in muscle mass of up to 50% in patients in their 80s [9]. The mechanisms that lead to sarcopenia are not entirely understood, but research has shown that muscle loss is affected by other external factors including hormone levels, steroids, and physical inactivity. Cachexia is a form of excessive weight loss with a disproportionate amount of muscle wasting that is linked to illnesses or disorders such as cancer, type I diabetes, and HIV. Over 5 million people in the USA suffer from cachexia [10]. This disorder has been linked to excess myostatin (which suppresses muscle growth) or glucocorticoids, or a deficiency of testosterone and insulin-like growth factor I (IGF-1) [10].

Both of these conditions are characterized by a loss of skeletal muscle mass and function [11,12,13,14,15]. At the cellular level, the failure of satellite cells or myoblasts to fuse together and form myofibers or decreases in their number and function are reported to play an important role in such disorders [16, 17]. The reduced regenerative capability of muscle is thought to be partially attributed to the decreased response of these cells to various growth factors and agents, or a decrease in the availability of sufficient growth factors that help with the maintenance and sustenance of muscle mass and satellite cells [2]. Among the potential causes for muscle wasting is the impairment of satellite cell proliferation, differentiation, or fusion of myoblasts into myofibers [2, 11,12,13,14,15, 18]. It is reported that in atrophied muscle tissue, the number of precursor cells, or the satellite cells, decrease over time, which results in defective myoblast fusion [14].

Muscle regeneration research with respect to satellite cells or myoblasts has stimulated interest in discovering factors that enhance proliferation and differentiation as the first step of the regenerative process. It has been observed that these cells respond well in vitro to different growth factors in a concentration-dependent manner [8]. Researchers have also noted the importance that nutrition plays in regulating muscle development and muscle protein synthesis at the tissue level [11]. Many natural compounds such as green tea [19], resveratrol [20], coffee [21], and vitamin D [22] have been used in vitro to enhance cell proliferation, differentiation, and fusion. One additional substance that could be a promising candidate for enhancing muscle development in vitro is fertilized chicken egg yolk as it contains nutrients such as vitamins, minerals, fats, amino acids, and proteins [23]. Specifically, chicken egg yolk contains cholesterol, vitamins (e.g., A, B1, B2, D), minerals (e.g., Ca, K, Mg), amino acids (aspartic acid, leucine, lysine) and proteins (e.g., Phosvitin).

The fertilized chicken egg yolk product Fortetropin® has been previously evaluated in a randomized, double-blind, placebo-controlled human clinical study in order to assess its impact on muscle gain in 18–21-year-old male subjects [21]. In that study, it was observed that subjects that consumed Fortetropin® on a daily basis while performing moderate resistance training activities twice per week experienced statistically significant gains in muscle mass relative to the macronutrient-matched placebo group. In a preclinical rodent study, Fortetropin® was found to upregulate anabolic signaling (mTor pathway activity), downregulate catabolic signaling (ubiquitin proteasome pathway activity), and downregulate gene expression of ActRIIB, the receptor for myostatin [21].

In the present study, we treated myoblast cultures with nutritional supplements to observe the effects of different concentrations of the supplement (fertilized egg yolk extract) on C2C12 myoblasts. Mitochondrial activity, fluorescence imaging, and mRNA expression of genes were analyzed at specific time points in order to observe the proliferation and differentiation stages of myoblasts. Our aims were twofold: (1) to assess effect of fertilized egg yolk extract on myoblast proliferation and growth and (2) to assess effect of fertilized egg yolk extract on myoblast fusion and differentiation.

Materials and Methods

Preparation of Egg Yolk Solution

Fertilized and unfertilized egg yolk (stored at − 20 °C) obtained from MYOS RENS Technology, Inc. (Cedar Knolls, NJ) was first frozen at − 80 °C and then pulverized into a powder using a mortar and pestle (stored at − 80 °C). When fertilized eggs are purchased from a local farm, the assumption is made that when hens and roosters are placed together in a pen in a defined ratio (i.e., 1:20), a very large proportion of the eggs will be fertilized based on historical experience/data. This was tested in a previous study using a total DNA content measurement to verify fertilization [24].

Both egg yolk powders were then dissolved in PBS in order to prepare a 50 mg/ml egg yolk stock solutions. This concentration was chosen because of the ease of getting this yolk into solution. The stock fertilized egg yolk solutions were then added to the media (described earlier) at concentrations of 500 μg/ml, 1 mg/ml, and 10 mg/ml to give a low, medium, and high concentration.

Cell Culture

C2C12 (ATCC) cells were expanded in Dulbecco modified eagle medium (DMEM) (Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) (Fisher Scientific) and 1% (v/v) penicillin and streptomycin (P/S) (Gibco® by Life Technologies). The antibiotics penicillin and streptomycin were added to the media to prevent microbial contamination. The cell line was maintained in the growth medium (DMEM+ 10%FBS+ 1%P/S) at 37 °C in 5% CO2 atmosphere. Cell culture studies were performed after approximately 80% cell confluence was attained. Cells were then seeded into 96-well plates with a cell density of 10,000 cells/cm2. The cell proliferation, differentiation, and myofiber formation were monitored daily until day 7 using a standard light microscope (Olympus CKX31).

After 7 days, cell culture studies were divided into two parts: (1) a proliferation study and (2) a differentiation study. For the proliferation study, media was replaced with fresh control growth media (DMEM+ 10%FBS+ 1%P/S) supplemented with either 500 μg/ml, 1 mg/ml, or 10 mg/ml fertilized egg yolk stock solution (day 0). Groups in this study are listed in Table 1, n = 4.

Table 1 Proliferation study groups (n = 4)

The differentiation study was similar to the proliferation study (Table 1), except that the media was changed from proliferation media to differentiation media (DMEM+ 2%FBS+ 1%P/S). The C2C12 cells showed no signs of trans-differentiation into adipocyte or other lineages when exposed to the egg yolk.

Cell Viability/Metabolism

Cell viability assays, Presto Blue® (Invitrogen) were performed on days 1, 3, and 7 during the cellular studies (n = 3). The assay measured mitochondrial activity which can be used as an indirect measure of cell viability. The basis of the assay was measuring a change in color (from blue to dark violet), measured in the form of fluorescence units. Presto Blue® solution was prepared by diluting the reagent (1:10) with media under dark conditions. Media was removed from all wells and the wells were then washed with phosphate buffered saline (PBS). The Presto Blue solution was then added to each well and incubated for 1 h in the dark. After the incubation period, Presto Blue® solution from each well was collected in a new well plate. The readings were measured using a microplate photometer (Infinite M200 Pro, Tecan) with the assistance of i-control software, version 1.9. Relative absorbance for every sample was calculated using the average values and standard deviation.

Cytochemical Analysis and Fluorescence Imaging

Cytochemical analysis was carried out directly on cells in the 96-well plates on day 7 (n = 3). Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) at 20 °C for 20 min. The cells were then washed again with PBS and stored at 4 °C overnight. Cells were treated with triton-X (0.5%) solution for 15 min, washed with PBS and then incubated with 1% bovine serum albumin (BSA) (Fisher Scientific) for 30 min. The cells were then stained with fluorescein phalloidin (1:20) (Life Technologies) for 20 min at R.T., washed with PBS, and counter stained with DAPI (Life Technologies) for 5 min.

All of the wells were imaged with a spinning disc confocal microscope (Olympus IX81) in order to visualize actin fibers and nuclei of cells at 20× objective. Slidebook 5 software (Intelligent Imaging Innovations, Denver, CO) was used to capture images of the samples. ImageJ software was used to analyze number of nuclei, number of myotubes, and fusion index. Fusion index was calculated using the formula: number of nuclei on myotubes/total number of nuclei [25].

Real-Time Quantitative PCR analysis

qPCR was performed on day 3 to analyze mRNA expression of the muscle-specific genes MyoD and myogenin in cells (n = 3). The cells were lysed using RLT Plus Buffer with β-mercaptoethanol (10 μl of β-mercaptoethanol for every 1 ml of RLT Plus Buffer) and RNA was extracted using the RNeasy Plus mini kit (Qiagen). Total RNA was quantified with a Nanodrop 2000c spectrophotometer (Thermo Scientific) and reverse transcribed to create cDNA library using high-capacity cDNA reverse transcription kit (Life Technologies) on a Peltier Thermal Cycler (PTC-200, MJ Research). The resultant cDNA was amplified using the primers (Harvard Medical School Primer Bank, https://pga.mgh.harvard.edu/primerbank/) listed in Table 2, several primers were used for MyoD (two) and myogenin (four) in order to find the primer that was most well suited [26,27,28,29]. PikoReal 96™ Real-Time PCR System (Thermo Fisher Scientific) was used to analyze the PCR products. qPCR measurement was performed in triplicate. The SYBR green technique was then used to detect the PCR products. The relative quantification of real-time PCR was carried out according to the method described in the literature [30, 31]. The values were normalized to a reference gene, GAPDH.

Table 2 qPCR primer sequences

Statistical Analysis

All the values were represented using the mean and standard deviation. One-way analysis of variance (ANOVA) was carried out using Synergy software by Kaleidagraph® to determine statistical significance. A value of p ≤ 0.10 was considered significant using Fischer’s least significant difference comparison. All the data are represented as mean ± standard deviation (± S.D).

Results

Growth Media: Cell Viability/Metabolism Studies

At 500 μg/ml, overall cell viability increased from day 1 to day 7 for all groups, Fig. 1. In the control group (no egg yolk supplement), there was a significant increase in metabolism with each increasing time point. In the egg yolk-supplemented groups, the cell metabolism decreased on day 3 and then increased on day 7 for groups supplemented with egg yolk (although only significant for unfertilized egg yolk). Cell metabolism on day 7 was significantly higher (p ≤ 0.05) in fertilized egg yolk-supplemented groups relative to the control group (Tables 3, 4, 5, and 6).

Fig. 1
figure1

Cell viability/metabolism measurements obtained using the Presto Blue Assay (n = 4). The control group is media with 10% fetal bovine serum (FBS), the unfertilized group is media with 10% FBS supplemented with 500 μg/ml unfertilized egg yolk extract, and the fertilized group is media with 10% FBS supplemented with 500 μg/ml fertilized egg yolk extract. Significant difference (p ≤ 0.05) between groups is represented by asterisk

Table 3 Image analysis using ImageJ for 500 μg/ml egg yolk-supplemented media (10%FBS). A significant difference (p ≤ 0.1) between control and groups is represented by asterisk
Table 4 Image analysis using ImageJ for 1 mg/ml and 10 mg/ml fertilized and unfertilized egg yolk-supplemented media (10%FBS). A significant difference (p ≤ 0.1) between groups is represented by asterisk
Table 5 Image analysis using ImageJ for 1 mg/ml and 10 mg/ml fertilized egg yolk-supplemented media (2%FBS). A significant difference (p ≤ 0.10) from control and 10 mg/ml unfertilized egg yolk are represented by *and # respectively
Table 6 Image analysis using ImageJ for 500 μg/ml fertilized egg yolk-supplemented media (2%FBS). Significance was not checked because of the small sample size n = 2

Similar to the myoblasts supplemented with 500 μg/ml egg yolk, myoblasts supplemented with 1 mg/ml egg yolk displayed an increase in viability/metabolism from day 1 to day 7, Fig. 2. Myoblasts supplemented with 10 mg/ml of egg yolk (either fertilized or unfertilized) displayed a continued increase in viability with each increasing timepoint (although not significant between days 3 and 7), Fig. 2. The control and 1 mg/ml egg yolk groups displayed a decrease in viability from day 3 to day 7 (although not significant). The 10 mg/ml groups displayed increased viability compared to control at day 7. The proliferative trends of myoblasts supplemented with 1 mg/ml are similar to those seen in the control myoblasts, significant increases from day 1 to day 3 followed by a decrease from day 3 to day 7 (not significant). In addition, there are no significant differences between the two groups at any time point.

Fig. 2
figure2

Cell viability/metabolism measurements obtained using the Presto Blue Assay (n = 4). The control group is media with 10% fetal bovine serum (FBS), U is supplemented with unfertilized egg yolk at the listed concentration, and F is supplemented with fertilized egg yolk at the listed concentration. Significant differences (p ≤ 0.05) between groups are represented by asterisk

Growth Media: Fluorescence Image Analysis

At a supplemented egg yolk concentration of 500 μg/ml, significant differences were not observed in the number of nuclei and the number of myotubes between any of the groups. However, a significant difference (p ≤ 0.05) was observed in fusion index between control media and the 500 μg/ml egg yolk-supplemented media (fertilized and unfertilized). There was no significant difference in fusion index between myoblasts supplemented with fertilized and unfertilized egg yolk (Figs. 3 and 4).

Fig. 3
figure3

Image analysis using ImageJ for 500 μg/ml egg yolk-supplemented media (10%FBS) including a number of nuclei, b number of myotubes, c number of nuclei per myotube, and d fusion index. A significant difference (p ≤ 0.1) between control and groups is represented by asterisk

Fig. 4
figure4

Fluorescence images of C2C12 myoblasts after 7 days under different growth culture conditions. Cells are stained with Phalloidin (green) for actin and DAPI (blue) for nuclei. The culture conditions are a control with no egg yolk, b 500 μg/ml unfertilized egg yolk, c 500 μg/ml fertilized egg yolk, d 1 mg/ml unfertilized egg yolk, e 1 mg/ml fertilized egg yolk, f 10 mg/ml unfertilized egg, and g 10 mg/ml fertilized egg. Scale bar = 400 μm

Cells were cultured in growth media with two higher concentrations of egg yolk: 1 mg/ml and 10 mg/ml. There were significant increases in the number of nuclei between both unfertilized egg yolk solutions and the control (p ≤ 0.05), and the fertilized egg yolk group at 10 mg/ml and the control. Both 10 mg/ml egg yolk groups also showed a significant increase in the number of myotubes compared to the control (Figs. 4 and 5). At 10 mg/ml, there was a significant increase in fusion index in fertilized egg yolk compared to unfertilized egg yolk.

Fig. 5
figure5

Image analysis using ImageJ for 1 mg/ml and 10 mg/ml egg yolk-supplemented media (10%FBS) including a number of nuclei, b number of myotubes, c number of nuclei per myotube, and d fusion index. A significant difference (p ≤ 0.1) between control and groups is represented by asterisk

Differentiation Media: Cell Viability/Metabolism Studies

After incubation in differentiation media, the cell viability/metabolism measured on day 3 and day 7 was significantly higher (p ≤ 0.05) for the 500 μg/ml egg yolk-supplemented media groups as compared to the control media group. Cell metabolism increased from day 1 to day 7 only for the 500 μg/ml egg yolk groups, while it decreased in the control (p ≤ 0.05) (Fig. 6). When cultured in 500 μg/ml egg yolk-supplemented media, myoblasts showed a significant increase in viability from day 3 to day 7.

Fig. 6
figure6

Cell viability using the Presto Blue Assay (n = 4) for C2C12 myoblasts cultured in media with 2% fetal bovine serum (FBS) supplemented with 500 μg/ml fertilized and unfertilized egg yolk extract A significant difference with the control group at that time point (p ≤ 0.10) is represented by asterisk. A significant difference between groups (p ≤ 0.05) is represented by #.

Following the experiments that were performed with media supplemented with 500 μg/ml of fertilized egg yolk solution, additional assays were performed. The concentration was varied (1 mg/ml and 10 mg/ml), and one control was used (Fig. 7). The cell metabolism decreased from day 1 to day 7 for control media and 10 mg/ml fertilized egg yolk-supplemented media while it increased from day 1 to day 7 for 1 mg/ml fertilized egg yolk-supplemented media and for both concentrations of unfertilized egg yolk (though not significantly). On day 7, the cell metabolism was significantly higher (p ≤ 0.05) for 1 mg/ml fertilized egg yolk, 10 mg/ml unfertilized egg yolk, and 1 mg/ml unfertilized egg yolk-supplemented media as compared to the control media (Fig. 7).

Fig. 7
figure7

Cell viability using the Presto Blue Assay (n = 4) for C2C12 myoblasts cultured in media with 2% fetal bovine serum (FBS) supplemented with 1 mg/ml and 10 mg/ml fertilized and unfertilized egg yolk extract. A significant difference with the control group at that time point (p ≤ 0.05) is represented by asterisk

mRNA expression of MyoD was higher for 500 μg/ml unfertilized egg yolk-supplemented group than control (p = 0.0628). MyoD expression was also higher for the 500 μg/ml unfertilized egg yolk-supplemented group compared to 10 mg/ml unfertilized egg yolk (p = 0.0498). MyoD expression was also higher for the 500 μg/ml and 1 mg/ml fertilized egg yolk-supplemented group compared to 10 mg/ml fertilized egg yolk (p = 0.0498 and p = 0.0882 respectively). Other trends in MyoD expression were an increase in expression in 500 μg/ml fertilized egg yolk and 1 mg/ml egg yolk (fertilized and unfertilized) compared to control; these differences were not significant (Fig. 8).

Fig. 8
figure8

The mRNA expression of MyoD on day 3. Media with 2% fetal bovine serum (FBS) supplemented with different concentrations of fertilized egg yolk extract. A significant difference (p ≤ 0.05) between groups is represented by asterisk

The mRNA expression of myogenin was significantly higher for 500 μg/ml egg yolk (fertilized and unfertilized) supplemented media than the control media (Fig. 9). Myogenin expression for the 1 mg/ml fertilized egg yolk was larger than expression for the control (p = 0.0514). Myogenin expression for 500 μg/ml and 1 mg/ml fertilized egg yolk were larger than expression for 10 mg/ml fertilized egg yolk (p = 0.0308 and p = 0.0911 respectively).

Fig. 9
figure9

The mRNA expression of Myogenin on day 3. Media with 2% fetal bovine serum (FBS) supplemented with different concentrations of fertilized egg yolk extract. A significant difference (p ≤ 0.05) between groups is represented by asterisk

Differentiation Media: Fluorescence Image Analysis

The C2C12 myoblasts were also subjected to higher doses (1 mg/ml and 10 mg/ml) of egg yolk (unfertilized and fertilized), Figs. 10 and 11. The 10 mg/ml and 1 mg/ml fertilized egg yolk group displayed an increase in nuclei count above the control group (p = 0.0122 and p = 0.0069). In addition, 1 mg/ml unfertilized egg yolk group displayed an increase in nuclei count above the control group (p = 0.0643). The 10 mg/ml unfertilized egg yolk group displayed an increase in myofiber count above the control group (p = 0.0714). At 10 mg/ml, the fusion index for fertilized egg yolk was higher than the index for 10 mg/ml unfertilized egg yolk (p = 0.0243).

Fig. 10
figure10

Image analysis using ImageJ for 1 mg/ml and 10 mg/ml egg yolk-supplemented media (10%FBS) including a number of nuclei, b number of myotubes, c number of nuclei per myotube, and d fusion index. A significant difference (p ≤ 0.1) from control and 10 mg/ml unfertilized egg yolk are represented by *and # respectively

Fig. 11
figure11

Fluorescence images of C2C12 myoblasts on day 7. Myogenic differentiation of C2C12 myoblasts in a control media, b 1 mg/ml fertilized egg yolk-supplemented media, and c 10 mg/ml fertilized egg yolk-supplemented media

Discussion

Compromised muscle regenerative capacity as observed in muscle wasting disorders can be due to the reduced number of myoblasts or myoblast dysfunction [32]. To address this issue, groups have explored muscle growth at the in vivo level, investigating nutritional intervention as a promising approach to promote muscle growth and development [33, 34]. One-way potential way to examine the utility of these nutritional interventions is to observe their effectiveness using the tools of regenerative and tissue engineering. As the opposite of muscle wasting is muscle growth, tools to measure muscle growth and development at the cellular level should be adequate in reviewing the effectiveness of these treatments. In the present study, we have studied the effect of fertilized egg yolk extract (a potential nutritional intervention) on the behavior and morphology of myoblasts under cell culture conditions that have been shown to promote proliferation and differentiation of C2C12 cell line, a commonly used model to study myogenesis in vitro [35]. Cellular metabolism, mRNA expression of myogenic genes, and morphology through fluorescence microscopy were utilized to determine whether supplementation of growth media with fertilized egg yolk affects the process of myogenesis.

One potential consideration for the use of egg yolk is the potential for allergies. Egg allergies are a very common food allergy in children; reactions include hives, abdominal pain, nasal congestion, and anaphylaxis [36]. Egg white allergies are more common than egg yolk allergies [36]. One study concluded that children who were allergic to heated hen’s egg were able to safely consume the heated yolk of the egg [37]. Although this does bode well for the use of fertilized egg yolk, the possibility of an allergic response should be considered when using fertilized egg yolk in vivo.

Effect of Egg Yolk on Myoblast Proliferation and Growth in Media Supplemented with 10% FBS and 2% FBS

Cellular metabolism/viability, increased from day 1 to day 7 for all groups, Fig. 1. The 500 μg/ml fertilized egg yolk-supplemented media group was significantly higher than the control on day 7. To confirm whether an increase in the concentration of egg yolk supplemented in media would change cell metabolism, higher concentrations of egg yolk (1 mg/ml and 10 mg/ml) were used. Myoblasts supplemented with 1 mg/ml and 10 mg/ml egg yolk displayed an increase in viability/metabolism from day 1 to day 7, Fig. 2. The 10 mg/ml groups displayed increased viability compared to control at day 7.

When switching to differentiation media at a concentration of 500 μg/ml egg yolk, supplemented media myoblasts showed a significant increase in viability from day 1 to day 7 while the control showed a significant decrease from 1 to 7. This is most likely due to the increased protein present in these groups compared to the control. For higher egg yolk concentrations, there was no increase in metabolism/viability, Fig. 8. On day 7, the cell metabolism was significantly higher for 1 mg/ml fertilized egg yolk, 10 mg/ml unfertilized egg yolk, and 1 mg/ml unfertilized egg yolk-supplemented media compared to the control. Once again, this is most likely due to the increased protein available to the cells.

Effect of Fertilized Egg Yolk on Myoblast Differentiation and Fusion in Media Supplemented with 10% FBS and 2% FBS

To determine how the fertilized egg yolk affects early stage myogenesis, mRNA expression of MyoD and myogenin was analyzed on day 3 since the fusion of myoblasts into myotubes were apparent on day 3. MyoD and myogenin are myogenic transcription factors for skeletal muscle which determine the fate of the myoblasts in their early differentiation stage. MyoD is an important transcription factor for muscle development [6]. When overexpressed, MyoD upregulates many myogenic genes (i.e., myogenin) resulting in the transdifferentiating of non-muscle cells into cells that produce muscle-like phenotypic characteristics [38]. Myogenin also plays a key role in the muscle development by regulating gene encoding of myosin and other muscle differentiating genes [39]. These are not the only factors that are expressed during the process of myogenesis but their expression would suggest that the myoblasts are fusing into myotubes. Increases in egg yolk concentration seemed to have an effect on MyoD expression. mRNA expression of MyoD was higher for 500 μg/ml unfertilized egg yolk-supplemented group than control (p = 0.0628) and 10 mg/ml unfertilized egg yolk (p = 0.0498). MyoD expression was also higher for the 500 μg/ml and 1 mg/ml fertilized egg yolk-supplemented group compared to 10 mg/ml fertilized egg yolk (p = 0.0498 and p = 0.0882 respectively). Myogenin expression displayed a similar concentration dependent increase and then decrease. mRNA expression of myogenin was higher for 500 μg/ml egg yolk (fertilized and unfertilized) supplemented media than the control media (Fig. 11). Myogenin expression for 1 mg/ml fertilized egg yolk supplemented cells was larger than control (p = 0.0514). Myogenin expression for 500 μg/ml and 1 mg/ml fertilized egg yolk was larger than that for 10 mg/ml egg yolk (p = 0.0308 and p = 0.0911 respectively).

At a supplemented egg yolk concentration of 500 μg/ml, the fusion index for the control group was significantly lower (p ≤ 0.05) than egg yolk-supplemented media (fertilized and unfertilized). At this concentration, there was no significant difference in fusion index between myoblasts supplemented with fertilized and unfertilized egg yolk (Figs. 3 and 4).

When the amount of egg yolk was increased, the egg yolk-supplemented groups displayed larger values than the control group in different categories. There were significant increases in the number of nuclei between both unfertilized egg yolk solutions and control, and the fertilized egg yolk group at 10 mg/ml and the control. Both 10 mg/ml egg yolk groups displayed more myotubes than control (Fig. 4). There was a difference between the egg yolk-supplemented groups at 10 mg/ml. At this concentration, there was a significant increase in fusion index in fertilized egg yolk compared to unfertilized egg yolk.

When the amount of egg yolk added to the differentiation media was increased, there were increases in different values comparing the supplemented groups to control. Similar to the growth media, at a concentration of 10 mg/ml, the fusion index for fertilized egg yolk was higher than the index for unfertilized egg yolk (p = 0.0243).

This data reveals that the addition of egg yolk to myoblasts does alter behavior, proliferative, and differentiation. These changes show some level of concentration dependence. There are increases over control with the addition of egg yolk for viability/metabolism. As expected, these changes are more profound in the differentiation media because of the decrease in available serum (2% compared to 10%). In this case, the increased protein from the egg yolk provides additional nutrients to support cell growth. Any changes in measures of differentiation seem to increase with low to medium concentrations of egg yolk (500 μg/ml and 1 mg/ml) and decrease at large concentrations (10 mg/ml). Differences between the types of egg yolk were only seen in measurements of myoblast differentiation (specifically fusion index) and only at the largest concentrations tested (10 mg/ml).

Conclusion

Overall, supplementation of fertilized egg yolk enhanced proliferation and differentiation of myoblasts in a dose-dependent manner. This was characterized by increased cell metabolism, number of nuclei, number of myotubes, fusion index, and mRNA expression of myogenin. Additional methods of analysis such as immunostaining (e.g., MHC, myogenin) and additional samples and time points are needed to potentially determine ideal concentration of fertilized egg yolk that can be supplemented to improve muscle generation in vitro.

Although this is a preliminary, in vitro study, this data highlights the potential benefits of fertilized egg yolk at a basic level to improve myogenesis. In order to further demonstrate its value as a nutritional product to improve myogenesis, additional studies must be performed including separating the fertilized egg yolk into more fundamental components and using animal models to complement any in vitro results.

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Acknowledgments

We thank MYOS RENS Technology, Inc., for supplying us with fertilized egg yolk extract and Dr. Richard Cohen (Rutgers University) for lending his expertise to the project.

Funding

This work was supported by MYOS RENS Technology, Inc., Cedar Knolls, NJ.

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Correspondence to Joseph W. Freeman.

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This work was financially supported by MYOS RENS Technology, Inc., Cedar Knolls, NJ.

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Joglekar, D., Warren, R., Browe, D. et al. Investigating the Effects of Fertilized Egg Yolk Extract on Myoblast Proliferation and Differentiation. Regen. Eng. Transl. Med. 6, 125–137 (2020). https://doi.org/10.1007/s40883-019-00137-y

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Keywords

  • Muscle wasting
  • Satellite cells
  • Myoblasts
  • Skeletal muscle regeneration
  • Nutrition
  • Fertilized egg yolk
  • C2C12 myoblasts