Effect of heparan sulfate and gold nanoparticles on muscle development during embryogenesis

Figures & data

Figure 1 Image of hydrocolloid of gold nanoparticles visualized using transmission electron microscopy.

Table 1 Oligonucleotide sequences used in polymerase chain reaction

Gene	Size	Primer sequences (5′ to 3′)
ACTB	ca. 169 bp	Forward: GTC CAC CTT CCA GCA GAT GT
		Reverse: ATA AAG CCA TGC CAA TCT CG
EEF1-α	ca. 85 bp	Forward: AGC AGA CTT TGT GAC CTT GCC
		Reverse: TGA CAT GAG ACA GAC GGT TGC
FGF-2	ca. 151 bp	Forward: GGC ACT GAA ATG TGC AAC AG
		Reverse: TCC AGG TCC AGT TTT TGG TC
PAX-7	ca. 135 bp	Forward: CCA GTA GAG ACA GGC CAA GC
		Reverse: GGA GTT GGG AAG GAG TAG GG

Table 2 Mortality and average weight of embryos and organs without (–) or with gold nanoparticles and heparan sulfate

	Treatment				ANOVA
	Without HS		With HS		Effect				Interaction
	–	Nano-Au	–	Nano-Au	Nano-Au		HS		P
					P	SEM	P	SEM
Mortality, %	13.6	15.6	9.52	8.33
Embryo, % e.w	85.1	84.9	85.0	85.0	0.615	0.29	0.917	0.26	0.045
Liver, % b.w	1.66	1.75	1.72	1.69	0.074	0.030	0.447	0.029	0.613
Heart, % b.w	0.60	0.59	0.63	0.56	0.347	0.011	0.002	0.013	0.099
Spleen, % b.w	0.05	0.04	0.05	0.04	0.066	0.002	0.094	0.002	0.844

Abbreviations: % e.w, % of egg weight; % b.w, % of body weight; ANOVA, analysis of variance; HS, heparan sulfate; nano-Au, gold nanoparticles; SEM, standard error of the mean.

Table 3 Biochemical indices in the blood serum of chicken embryos without (–) or with gold nanoparticles and heparan sulfate

	Treatment				ANOVA
	Without HS		With HS		Effect				Interaction
	–	Nano-Au	–	Nano-Au	Nano-Au		HS		P
					P	SEM	P	SEM
Magnesium (mmol/L)	0.75	0.88	0.75	0.88	0.702	0.516	0.477	0.129	0.882
Calcium (mmol/L)	2.70	2.71	2.45	2.96	0.980	0.136	0.092	0.038	0.752
Phosphorus (mmol/L)	1.73	1.69	1.71	1.70	0.651	0.060	0.916	0.059	0.751
Triglycerides (mmol/L)	0.78	0.75	0.79	0.73	0.714	0.062	0.529	0.062	0.962
Cholesterol-VLDL (mmol/L)	0.35	0.35	0.36	0.34	0.904	0.039	0.740	0.036	0.928
Glucose (mmol/L)	10.6	10.2	10.1	10.2	0.502	0.51	0.689	0.507	0.899
Alkaline phosphatase (U/L)	658	636	567	727	0.812	64.2	0.093	54.2	0.620
Aspartate aminotransferase (U/L)	144	162	181	125	0.512	19.2	0.050	29.2	0.138
Alanine aminotransferase (U/L)	11.7	10.2	13.2	8.70	0.527	1.707	0.075	1.88	0.839
Lactate dehydrogenase (U/L)	2720	3047	2945	2822	0.223	184.2	0.643	192.3	0.399

Abbreviations: ANOVA, analysis of variance; HS, heparan sulfate; nano-Au, gold nanoparticles; SEM, standard error of the mean; VLDL, very-low-density lipoprotein.

Table 4 Morphometry of the breast muscle of chicken embryos without (–) or with gold nanoparticles and heparan sulfate

	Treatment				ANOVA
	Without HS		With HS		Effect				Interaction
	–	Nano-Au	–	Nano-Au	Nano-Au		HS		P
					P	SEM	P	SEM
Number of myocytes/10,000 μm^2*	23.0	27.5	18.5	32.1	0.197	3.21	0.0000	2.88	0.024
Number of satellite cells/10,000 μm^2*	7.1	8.4	6.8	8.7	0.213	0.91	0.0330	0.72	0.452
Fiber area (μm^2)*	19.9	25.0	19.2	21.4	0.002	1.06	0.0007	1.30	0.023
Number of nuclei/10,000 μm^2**	16.1	19.7	15.7	21.4	0.004	0.84	0.0009	1.03	0.0009

* Notes: Transmission electron microscope;

** confocal microscope.

Abbreviations: ANOVA, analysis of variance; HS, heparan sulfate; nano-Au, gold nanoparticles; SEM, standard error of the mean.

Figure 2 Images of nuclei (blue), stained with 4′,6-diamidino-2-phenylindole, in the cross-section of the breast muscle of a chicken embryo on day 20 of incubation visualized using a confocal microscope. (A) Control, (B) gold nanoparticles, (C) heparan sulfate, and (D) gold nanoparticles and heparan sulfate complex.

Figure 3 Images of the ultrastructure of the cross-section of the breast muscle of a chicken embryo on day 20 of incubation visualized using scanning electron microscopy (environmental scanning electron microscope mode). (A) Control, (B) gold nanoparticles, (C) heparan sulfate, and (D) gold nanoparticles and heparan sulfate complex.

Figure 4 Images of the ultrastructure of the cross-section of the breast muscle of a chicken embryo on day 20 of incubation visualized using transmission electron microscopy. (A) Control, (B) gold nanoparticles, (C) heparan sulfate, and (D) gold nanoparticles and heparan sulfate complex.

Figure 5 Images of Pax-7 positive nuclei, shown as an overlaid image of 4′,6-diamidino-2-phenylindole-stained nuclei (blue) and Pax-7 nuclei stained with fluorescein isothiocyanate (green), in the cross-section of the breast muscle of a chicken embryo on day 20 of incubation visualized using a confocal microscope. (A) Control, (B) gold nanoparticles, (C) heparan sulfate, and (D) gold nanoparticles and heparan sulfate complex.

Table 5 Expression of PAX-7 and FGF-2 on the messenger ribonucleic acid level, normalized to ACTB, and on the protein level in the breast muscle of chicken embryos without (–) or with gold nanoparticles and heparan sulfate

	Treatment				ANOVA
	Without HS		HS		Effect				Interaction
	–	Nano-Au	–	Nano-Au	Nano-Au		HS		P
					P	SEM	P	SEM
PAX-7*	0.80	0.98	0.57	1.21	0.435	0.156	0.0086	0.358	0.845
FGF-2*	2.63	2.70	4.40	0.94	0.950	0.705	0.0024	0.117	0.806
FGF-2 pg/mg protein**	132	127	144	118	0.716	8.4	0.217	10.3	0.119

* Notes: Messenger ribonucleic acid level;

** protein level.

Abbreviations: ANOVA, analysis of variance; HS, heparan sulfate; nano-Au, gold nanoparticles; SEM, standard error of the mean.