Increased nanosphere size in the cuticle layer of Japanese quail egg by mutation in the myostatin gene

Abstract

Cuticle quality can affect food safety by protecting poultry eggs from bacterial infection in the modern poultry industry. However, genetic factors related to cuticle nanostructure are not much reported due to limited bird models. In the current study, the genome-edited quail targeting myostatin (MSTN) gene was used to investigate the effect of MSTN mutation on the cuticle nanostructure and quality. To analyze nanostructure of the cuticle layer of the MSTN mutant and wild-type (WT) quail eggs, scanning electron microscope (SEM) images was taken. Thickness of the cuticle layer did not differ between the MSTN mutant and WT groups, but the size of the nanospheres in the surface of the cuticle layer was increased by MSTN mutation. In addition, increased size of the nanospheres in the MSTN mutant group was also shown in the upper region of the cross-sectional cuticle layer. Notably, both groups showed similar small-sized nanospheres in the lower region of the cuticle layer and the size was increased as they ascended to the upper region. The data suggested that MSTN mutation increased the size of the nanosphere in the upper region of the cuticle layer at a late phase rather than increasing the size of nanospheres in the lower region of the cuticle layer at an early phase of cuticle formation. However, the number of Escherichia coli attached to the surface did not differ between the two groups indicating no association between nanosphere size and bacterial attachment in quail eggs. The current study demonstrated a new function of the MSTN gene on regulation of cuticle nanostructure, for the first time. These results advanced our knowledge on the association between genetic factors and cuticle nanostructure and can be served as a reference to study the mechanism of cuticle formation in the future study.

Introduction

Avian eggshell is structurally sturdy and yet its quality is easily affected by various factors such as age, nutrition, and environment^1–4. Avian eggshell consists of different layers, the inner and outer eggshell membranes inside of the eggshell, the mammillary layer on the membrane, the thickest palisade layer in the middle, the thin vertical crystal layer, and the outermost cuticle layer⁵. Although the cuticle layer is absent in birds like the pigeon, budgerigar, munia, and canary⁶, the cuticle layer plays a critical role in protecting the eggs from external threats as a first barrier meeting the external environment directly. In addition to the effect of external factors such as ages and housing systems on cuticle deposition^7,8, nanostructure of the cuticle layer, presence or absence of nanospheres, can also affected by environmental factors such as humid or dry places⁹.

Among external threats, bacterial infection is a great risk in embryo viability. The presence of nanospheres in the cuticle layer shows an advantage in the protection of eggs from bacterial penetration by effectively clogging the pore canals¹⁰. Eggs of major poultry species, including chickens, turkeys, ducks, and quail, contain nanospheres in the cuticle layer⁶. They originally laid eggs on the ground where soil bacteria are abundant, and they might evolve to have nanospheres in the cuticle layer of their eggs to protect embryos from bacterial infection. Although soil bacteria are no longer causing a problem in the modern layer industry, the cuticle layer is still critical in food safety by protecting eggs from pathogens, such as Salmonella¹¹. In addition to the external factors affecting cuticle quality, therefore, genetic factors need to be also investigated to improve cuticle quality to secure egg safety in the layer industry.

Although genes involved in the eggshell biomineralization process have been identified¹², genetic factors affecting cuticle quality and structures are not fully understood, due to the absence of avian models. The anti-myogenic function of the myostatin (MSTN) gene, first discovered in knockout mice¹³, was demonstrated in genome-edited quail¹⁴ and chickens¹⁵. As an autosomal recessive gene, homozygous mutation in the MSTN gene resulted in increased body weight and muscle mass in male and female quail but not in heterozygous mutant quail¹⁴. In addition to the well-investigated function of the MSTN gene on muscle mass, the MSTN mutant quail was further used as an avian model to investigate the effect of MSTN mutation on egg laying performance¹⁶ and whole eggshell quality¹⁷. The MSTN mutant eggs were bigger but had thinner and weaker eggshells compared to wild-type (WT) eggs in quail¹⁷. These negative effects of the MSTN mutation on whole eggshell thickness and strength were caused by decreased thickness of the palisade layer, the thickness layer in the avian eggshell¹⁷. However, the effect of MSTN mutation on the cuticle layer specifically has not been investigated yet. In the current study, eggs of MSTN mutant and WT quail were collected to compare the nanostructure of the cuticle layer. Scanning electron microscopy was used to analyze nanosphere size of the surface and cross-section of the cuticle layer. In addition, bacterial attachment on the eggshell surface was compared between the two groups.

Results

Effect of MSTN mutation in the cuticle layer thickness of quail eggs

Eggshells of the MSTN mutant quail eggs were thinner than those of WT quail eggs (Fig. 1A), as reported in our previous study¹⁷. However, the thickness of the cuticle layer was similar between the two groups (Fig. 1B, C) P = 0.85).

Figure 1.

Thicknesses of the eggshell and cuticle layer of the myostatin (MSTN) mutant and wild-type (WT) quail groups. (A) Cross-sectional scanning electron microscope (SEM) images of whole eggshells. (B) Enlarged SEM images of the cuticle layer. Scale bar, 10 μm. (C) Comparison of the thickness of the cuticle layer between the two groups. n = 10 per group. The values represent the mean ± standard error of the mean.

Effect of MSTN mutation in the nanosphere size of the cuticle layer

Surface SEM images of the cuticle layer showed bigger nanospheres in the MSTN mutant group compared to the WT group (Fig. 2; P < 0.05). In a cross-section of the cuticle layer, nanospheres in the upper region of the cuticle layer were bigger than those in the lower region of the cuticle layer in both groups (Fig. 3; P < 0.05). Although the size of nanospheres in the lower region of the cuticle layer was not different between the two groups, nanospheres in the upper region of the cuticle layer of the MSTN mutant eggs were bigger than those of the WT eggs (Fig. 3; P < 0.05).

Figure 2.

Nanospheres on the surface of the cuticle layers of the MSTN mutant and WT quail groups. (A) SEM image showing nanospheres on the surficial cuticle layer of MSTN mutant quail egg. (B) SEM image showing nanospheres on the surficial cuticle layer of WT quail egg. Scale bar, 1 μm. (C) Comparison of diameters of the nanospheres between the two groups. n = 10 per group. The values represent the mean ± standard error of the mean, *P < 0.05.

Figure 3.

Nanospheres on the cross-section of the cuticle layers of the MSTN mutant and WT quail groups. (A) SEM image showing nanospheres in the cross-sectional cuticle layer of MSTN mutant quail egg. (B) SEM image showing nanospheres in the cross-sectional cuticle layer of WT quail egg. Scale bar, 1 μm. (C) Comparison of diameters of the nanospheres in the upper and lower regions of the cuticle layer between the two groups. n = 10 per group. The values represent the mean ± standard error of the mean. ^a–cMeans sharing the same superscript are not significantly different from each other, P < 0.05. (D) Enlarged SEM image showing nanospheres in the cross-sectional cuticle layer of MSTN mutant quail egg, Scale bar, 1 μm.

Effect of nanosphere size in bacterial attachment on the cuticle surface

Due to differences in sizes of the surficial nanospheres without changes throughout the cuticle layer, bacterial attachment on the surface of the cuticle layer was compared in the current study. The number of Escherichia coli cells attached to the surface of randomly selected areas did not significantly differ between the two groups (Fig. 4; P = 0.82).

Figure 4.

Comparison of bacterial attachment on the surface of the cuticle layers between the MSTN mutant and WT quail. (A) SEM image showing Escherichia coli attached to the surface of MSTN mutant quail egg. (B) SEM image showing Escherichia coli on the surface of WT quail egg. Scale bar, 5 μm. (C) Cell count of Escherichia coli attached to eggshell after 3 h of incubation. n = 6 per group. The values represent the mean ± standard error of the mean.

Discussion

The mechanism of nanosphere formation in the cuticle layer of the avian eggshell is not fully understood, although the biomineralization process of other eggshell layers have been investigated⁵. Unlike reduced thickness of the palisade layer¹⁷, similar thickness of the cuticle layer between the MSTN mutant and WT quail eggshells (Fig. 1) suggests a different effect of the MSTN mutation on formation of the inorganic mineralized palisade layer and the organic cuticle layer in the avian eggshell. Because eggshell formation takes the majority of time, almost 20 h within 24 h of the egg laying period in chickens⁵, longer egg laying cycle in the MSTN mutant quail compared to WT quail¹⁶ might favor in eggshell mineralization. However, reduction in thickness of the palisade layer without changes in cuticle layer thickness by the MSTN mutation might be due to differences in rate and/or duration of mineralization of the palisade layer. Formation of both palisade and cuticle layers occurs in the specific location of the oviduct called uterus, also known as shell gland^5,18. In addition to the major expression of MSTN in skeletal muscle¹³, MSTN is also expressed in the uterus of chickens¹⁹. More importantly, the expression level of MSTN was regulated by egg laying, as shown in a lower MSTN expression level in the uterus of egg-laying chickens compared to that of non-laying chickens at the same age¹⁹. In addition, the expression of follistatin, a strong inhibitor of MSTN, in the eggshell gland was increased toward a later phase of eggshell formation¹⁹, further suggesting that temporal regulation of MSTN activity might be important in eggshell formation.

The surficial and the upper region of the cuticle showed increased nanosphere sizes by MSTN mutation (Figs. 2 and 3). However, the size of nanospheres in the lower region of the cuticle layer were similar between the two groups indicating that the effect of MSTN mutation did not occur in the lower region of the cuticle layer where nanospheres are still small. The result showing the difference in nanospheres between the upper and lower regions in both groups indicates that nanospheres are getting bigger as they are deposited in the upper region of the cuticle layer. Based on the cross-sectional SEM image showing very small nanoparticles attached to the nanospheres (Fig. 3A and B), it is plausible that nanospheres are formed by coalescence of the very small nanoparticles. Especially, the traces of the very small nanoparticles in the nanospheres as shown in the enlarged SEM image (Fig. 3D) further confirmed that nanoparticles constituent the nanospheres. Although the mechanism of coalescence of very small nanoparticles into nanospheres needs to be investigated, increased size of nanospheres in the upper region suggests that the coalescence of the nanoparticles to make large nanospheres is positively affected by MSTN mutation.

One of the major functions of the cuticle layer is protecting against bacterial infection, and nanospheres could be optimally structured to block the pore canals against bacterial penetration yet allow gas exchange¹⁰. As an initial step of bacterial infection, bacterial attachment on the surface of the eggshell is affected by cuticle qualities such as surface hydrophobicity and nanosphere connectivity²⁰. Despite the different surficial nanosphere size, however, the numbers of Escherichia coli cells attached to the cuticle surface did not differ between the two groups, indicating that a small but significant difference in nanosphere size between WT and MSTN mutant eggs may not affect bacterial attachment. In addition, bacterial penetration rate is also expected to be similar between the two groups as the size of nanospheres in the lower region of the cuticle layer, where clogging of the pore canals occurs, did not differ between the two groups (Fig. 3). Indeed, similar hatchability between the two groups was reported in our previous study¹⁶, further supporting that there was no adverse effect on antimicrobial efficacy by increasing nanospheres in the cuticle layer by the MSTN mutation in quail. Instead, because a novel function of nanospheres on the surface of the eggshell as shock absorbers has been proposed in a tropical cuckoo²¹, the large nanospheres on the upper region of the cuticle layer in the MSTN mutant eggshell might have a beneficial effect on preventing the thin eggshell from cracks by external shock. Although potential consequence of increased size of nanospheres in the upper region of the cuticle layer need to be further investigated, the results will provide insight into understanding of the genetic factor on cuticle nanostructure and nanosphere formation.

In conclusion, A new function of the MSTN gene on regulation of cuticle nanostructure was demonstrated in the study by showing a bigger nanosphere in the surficial and upper region of the cuticle layer in the MSTN mutant quail eggs compared to those in the WT quail eggs. In addition, it is found that there is no association between nanosphere size and bacterial attachment. Improvement in eggshell quality, especially cuticle quality, is important in preventing eggs from bacterial infection for food safety and security. It is therefore important to advance our knowledge on the mechanisms of cuticle formation affected by genetic factors using genome-edited poultry species as a model.

Methods

Animal care

After a generation of genome-edited Japanese quail (Coturnix japonica) targeting the MSTN gene in our previous study¹⁴, the MSTN mutant and WT quail were maintained with ad libitum feeding at The Ohio State University Poultry Facility in Columbus, Ohio. Animal care protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee at The Ohio State University (Protocol 2019A00000024-R1; Approved January 21, 2022) and performed in accordance with the relevance guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.

Egg collection and preparation for scanning electron microscopy of cuticle layer

At 3.5 months old, female MSTN mutant and WT Japanese quail were transferred to individual cages and adapted to the circumstances for 2 weeks. Individual eggs were collected twice from 10 MSTN mutant and 10 WT quail to take scanning electron microscope (SEM) images of the eggshell surface to analyze nanospheres on the surface of the cuticle layer. Next, 10 MSTN mutant and 10 WT Japanese quail were prepared identically to obtain eggs for cross-sectional SEM images of the cuticle layers. To prepare eggshells for SEM image, eggs were broken vertically and soaked into distilled water for hours to rinse shells and remove membranes inside. The wet eggshells were dried overnight, and small pieces (0.5 × 0.5 cm) were taken from the equatorial region. Then, samples were mounted horizontally and vertically on aluminum stubes using carbon paint to analyze the surface and cross-section of the cuticle layer, respectively. After drying the paint overnight and sputter-coating of the samples with gold for 30 s at a current of 15 mA, SEM images were acquired using a Thermo Scientific Apreo LoVac Analytical Scanning Electron Microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA). The microscope was operated in its “high vacuum” mode at an accelerating voltage of 5.00 kV and current of 25 pA.

Preparation for scanning electron microscopy of bacteria attached eggshell

Eggs were collected from 6 MSTN mutant and 6 WT quail, and small pieces of eggshell (0.5 × 0.5 cm) from the equatorial region were prepared. After growing Escherichia coli with Tryptic Soy Broth, all samples were immersed into suspensions of the same concentration of Escherichia coli at room temperature for 3 h with slow shaking. Then, samples were fixed with 2.5% glutaraldehyde for overnight at 4 °C. After washing with PBS two times for 5 min, the samples were dehydrated in 70%, 85%, 100%, and 100% ethanol for 10 min, respectively. The dried samples were mounted on aluminum stubes using carbon paint. After sputter-coating of the samples with gold for 30 s at a current of 15 mA, a SEM image was taken using the Thermo Scientific Apreo LoVac Analytical Scanning Electron Microscope at an accelerating voltage of 10.00 kV and current of 1.6 nA.

Analysis of nanosphere size on the surface and cross-section of the cuticle layer and bacterial attachment on the cuticle surface

For all analysis, two SEM images were taken from different regions of each egg, and the measurements from the two images were averaged. To compare the nanosphere size on the surface of the cuticle layer between the groups, 100 nanospheres were randomly selected from each image, and diameter lengths of the nanospheres were measured using NIH ImageJ software (Ver. 1.52, http://imagej.nih.gov/ij). In the cross-sectional SEM images, areas within 2 μm below from the top and 2 μm above from the bottom were set as upper and lower regions of the cuticle layer, respectively. From each region, diameters of randomly selected 50 nanospheres were measured using NIH ImageJ software. Last, the number of bacteria attached to the surface of the cuticle layer were compared between the groups by manually counting them in the images.

Statistical analysis

A T test was performed to verify significant difference of the thickness, surficial nanosphere size, and number of bacterial attachments between the two groups using the SAS 9.4 software package (SAS Institute, Cary, NC). Cross-sectional nanosphere size in the upper and lower regions of the cuticle layer of the two groups were statistically analyzed using the GLM procedure. Duncan's Multiple Range comparison was used to evaluate and compare the variances between means of cross-sectional nanosphere size. Significance for all statistical tests were set at P < 0.05.

Author contributions

J.L., W.C., D.-H.K., C.M. and C.C. conducted the experimental works. J.L., W.C. and D.-H.K. wrote an original draft of the manuscript. J.L. and K.L. revised the manuscript. J.H., W.K. and K.L. supervised the study and provided guidance. All authors reviewed the manuscript.

Data availability

The data used during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.