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Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver Feng Gao a,∗ , Yingchun Liu b , Lingyao Li a , Ming Li a , Chongzhi Zhang a , Changjin Ao a , Xianzhi Hou a a b
College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 25 April 2014 Accepted 26 April 2014 Available online xxx
Keywords: Fetal growth restriction Maternal undernutrition Hepatic development Anti-oxidation capability Liver function
a b s t r a c t This study investigated the effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Eighteen ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.175 MJ ME kg BW−0.75 d−1 , n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW−0.75 d−1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 , n = 6). Fetuses were recovered at slaughter on d 140. Fetuses in the RG1 group exhibited decreased (P < 0.05) liver weight, total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), cholinesterase (CHE), total protein (TP), globulin (GLB), and alanine transaminase (ALT). In addition, intermediate changes were found in the RG2 fetuses, including decreased liver weight, T-AOC and CHE (P < 0.05). In contrast, increases in fetal hepatic collagen fibers and reticular fibers, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOs), monoamine oxidase (MAO), albumin (ALB)/GLB, aspartate transaminase (AST), and AST/ALT were found in the RG1 fetuses (P < 0.05). The RG2 fetuses had increased fetal hepatic collagen fibers, NOs and MAO (P < 0.05) relative to the control fetuses. These results indicate that impaired fetal hepatic growth, fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Intrauterine growth restriction (IUGR), often resulting from maternal undernutrition during pregnancy (Robinson et al., 1999; McMillen et al., 2001), is a significant cause of fetal and neonatal mortality and morbidity (Bernstein et al., 2000; Wu et al., 2006). Considerable epidemiological data indicate that the growth of the heart (Han et al., 2004; Bubb et al., 2007), lungs (Lipsett et al., 2006), and pancreas
∗ Corresponding author at: College of Animal Science, Inner Mongolia Agricultural University, No. 306# Zhao Wu Da Street, Hohhot 010018, Inner Mongolia, China. Tel.: +86 471 4309175; fax: +86 471 4301530. E-mail address: [email protected] (F. Gao).
(Dumortier et al., 2007) in IUGR fetuses are retarded, and these organs experience structural changes and impaired function that strongly predispose the animals to the development of cardiovascular disease (Barker, 1997), respiratory illness (Maritz et al., 2004), diabetes and insulin resistance (Vuguin et al., 2004; Green et al., 2010) in later life. As the largest of the body’s organs with the greatest number of functions, the developing fetal liver is sensitive to damage from both internal and external sources, including teratogens, infection and nutritional deficiencies (Hyatt et al., 2008). The impaired growth of fetal livers caused by maternal undernutrition has been observed (Bauer et al., 1995; Osgerby et al., 2002; Hyatt et al., 2002; Gao et al., 2009), and altered gene expression of the hepatic PRLGH-IGF axis has been detected in prenatal and postnatal
http://dx.doi.org/10.1016/j.anireprosci.2014.04.012 0378-4320/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Gao, F., et al., Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.04.012
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animals (Bauer et al., 1995; Hyatt et al., 2002, 2004, 2007). Although these data indicate that exposure to an adverse nutritional environment in pregnancy has a long-term impact on fetal or neonatal liver growth, additional studies are needed to ascertain whether reduced liver mass and the expression of growth factors in IUGR fetal sheep affect liver function and, indeed, whether such responses contribute to the developmental programming of poor health in later life (Hyatt et al., 2008). Therefore, the objective of the present study was to test the hypothesis that maternal undernutrition during late pregnancy impairs hepatic growth and is associated with liver dysfunction in IUGR ovine fetuses. 2. Materials and methods
Table 1 Composition of grass hay and the leftover hay during restriction period.
ME (MJ/Kg)a DM (%) CP (%) EE (%) NDF (%) ADF (%) ASH (%) Ca (%) P (%)
Grass hay
Leftover hay
8.90 88.42 10.09 4.34 71.98 35.82 4.67 0.57 0.09
– 91.99 9.27 2.72 71.19 36.60 4.39 0.68 0.08
a ME, metabolizable energy; DM, dry matter; CP, crude protein; NDF, neutraldetergent fiber; ADF, acid detergent fiber, EE = ether extract; Ca = calcium; P = phosphorus.
2.1. Animals and treatments All experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals in China (The State Science and Technology Commission of China, 1988). This study is a companion study, and the details of animals, experimental design and detailed procedures have been presented previously (Gao et al., 2013). Briefly, when the maternal nutrition density during late pregnancy is lower than the ensured “threshold” level (0.33 MJ ME kg BW−0.75 d−1 ), it may lead to Mongolian ovine fetal pathological changes and the ability of compensatory growth of postnatal offspring is suppressed or even lost (Gao, 2006; Qi, 2007; Gao et al., 2007, 2008, 2009). According to the previous results, three groups comprising eighteen Mongolian ewes carrying singletons were allocated at d 90 of pregnancy: a severely restricted group (Restricted Group 1: RG1, 0.175 MJ ME kg BW−0.75 d−1 , n = 6), a restricted group at the “threshold” level (Restricted Group 2: RG2, 0.33 MJ ME kg BW−0.75 d−1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 , n = 6). Second or third parity ewes were mated at synchronized estrus and had similar live weights (mean 52.82 ± 2.67 kg) during the d 90 of pregnancy. Pregnancies were confirmed by ultrasound scanning at approximately d 50 of gestation (Medison-SA-600, Shanghai, China). All animals were housed in individual pens, and chopped hay (mainly Leymus chinensis) was supplied ad libitum until d 90 of gestation. Based on the fact that the fetus is considered to achieve 80% to 85% of its final birth weight during the last two months of gestation (Robinson et al., 1999; Symonds et al., 2001), maternal undernutrition was imposed from d 90 to d 140 of pregnancy. At the beginning of restriction, the ME and chemical composition in the hay were measured (Table 1), and then the daily intake of the hay offered in RG1 and RG2 was calculated by the ewe body weight, nutrition value of hay, and the designed energy plane in the restricted groups. Restricted ewes were fed at 08:30 and 16:00 h each day, and the amount of feed offered was constant throughout the restriction period (Table 2). The ewes in the Control Group were offered feed at 08:30, 11:00 and 16:00 h daily (the feed refusals were approximately 10% of the total amount offered). The animals had free access to water and mineral mixture blocks (containing per kilogram: Ca, 15 g; P, 11.5 g; Mg as MgSO4 ·H2 O, 1 g; Fe as FeSO4 ·7H2 O, 500 mg;
Cu as CuSO4 ·5H2 O, 250 mg; Zn as ZnSO4 , 175 mg; Mn as MnSO4 , 100 mg; Co as CoCl2 ·6H2 O, 20 mg; I as KI, 40 mg; Se as Na2 SeO3 ·5H2 O, 1.5 mg; Yuantongweiye Co., Ltd., Inner Mongolian, China). All feed refusals were collected daily before feeding at 08:30, weighed and sub-sampled for chemical analysis.
2.2. Slaughtering procedures All fetuses were removed at d 140 of gestation. The umbilical cord blood was collected, and the fetal BW and liver weights were recorded. Some of the liver tissue was snap-frozen in liquid nitrogen and stored at −80 ◦ C. Tissue samples (approximately 1.0 cm3 ) were harvested from the large lobes of the liver. After rinsing with phosphatebuffered saline (PBS, pH 7.4), portions of the fetal liver were immediately placed in paraformaldehyde fixative solution (0.1 mol L−1 7.4). After fixation for at least 2 days, the tissues were dehydrated and paraffin-embedded, sectioned at 4–6 m and stained with commercial kits of reticulin stain (D032, NJJCBIO, Nanjing, China) and Masson stain (D026, NJJCBIO, Nanjing, China) for microscopic examination. Thereafter, five images were obtained from each section. Each specimen was viewed under a standard microscope, and the total number of collagen fibers and reticular fibers were counted in 10 random high power fields (HPF, magnification 100×) by three observers who were blinded to the study groups and results. Data were expressed as the number of collagen fibers per field and reticular fibers per field.
2.3. Liver chemical components analyses Fetal liver moisture was determined by freeze-drying to a constant weight (Christ, Alpha 1-4 lsc, German), and sample components were analyzed for chemical fat, protein and ash. The crude protein content was determined using the Kjeldahl method, and the values were converted to protein using the factor 6.25. The chemical fat content was determined as the difference in DM before and after extraction using ether. The ash content of the fetal liver was the residue left after ashing at 550 ◦ C in a muffle furnace.
Please cite this article in press as: Gao, F., et al., Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.04.012
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Table 2 Planes of maternal nutrition in different groups during late pregnancy. Nutrition levels
CG (ad libitum)a
RG2
RG1
Mean daily grass intake (g/d)b Mean daily crude protein intake (g/d) Daily metabolizable energy intakec (MJ ME kg BW−0.75 d−1 )
1689.05 170.43 0.670
842.49 85.01 0.330
440.18 44.41 0.175
a b c
CG = control group; RG2 = restricted group 2; RG1 = restricted group 1. Mean daily grass intake and crude protein intake are represented on a natural basis. Daily etabolizable energy intake is represented on a dry matter basis.
2.4. T-AOC, GSH-Px, SOD, and MDA in fetal livers
2.7. Statistical analysis
Approximately 0.5 g of the fetal liver was rinsed and homogenized in 0.85% chilled normal saline to obtain a 10% liver homogenate for the measurement of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. The activities of GSH-Px (A005) and SOD (A001-1), and MDA (A003-1) content were determined spectrophotometrically using commercial kits (NJJCBIO, Nanjing, China) according to the procedures of Paglia and Valentine (1967), Panckenko et al. (1975), and Placer et al. (1966), respectively. The total antioxidant capacity was determined using a spectrometric commercial kit (A015, NJJCBIO, Nanjing, China). In the reaction mixture, ferric ion was reduced by antioxidant reducing agents, and the blue complex Fe2+ -TPTZ (2,4,6tri (2-pyridyl)-s-triazine) was produced; absorbance was measured at 520 nm. One unit of T-AOC was defined as the amount that increased the absorbance by 0.01 at 37 ◦ C and was expressed as units per milliliter in the tissue homogenate.
All data were analyzed by using the ANOVA procedure as implemented in SAS software (the SAS Institute Inc, 2001). The model was as follows: Yi = u + Mi + ei , where u is the overall mean. Mi is the fixed effect of the nutrition treatments (i = 1–3), and ei is the random residual error. Duncan’s test was used to identify significant differences between mean values. Significance was declared at P ≤ 0.05.
2.5. NO, NOs, CHE and MAO in fetal livers A 10% liver homogenate was used to analyze the concentrations of the nitric oxide (NO, A013-1), nitric oxide synthase (NOs, A014-2), cholinesterase (CHE, 20120914), and monoamine oxidase (MAO, 20120914) in fetal livers according to the procedures of commercial kits (NJJCBIO, Nanjing, China). Each measurement was performed in duplicate.
3. Results 3.1. Fetal liver weights, structures and chemical component The effects of maternal undernutrition on the fetal liver weights, structures and chemical components are presented in Table 3 and Fig. 1. Fetal BW (P = 0.0001), liver weights (P = 0.0004) and fetal liver water content (P = 0.0014) in the restricted groups were reduced compared with the CG group, and there were differences in the fetal BW, liver weights and water content between the RG1 and RG2 groups (P < 0.05). In addition, the decreased protein, fat and ash components in fetal livers and a reduced fetal liver to brain weight ratio were found in RG1 as compared with the controls (P < 0.05). With the reduction of maternal energy intake, the number of fetal hepatic collagen fibers and reticular fibers per HPF in the RG1 and RG2 groups was increased, and there were differences in the number of collagen fibers among the RG1, RG2 and CG groups (P < 0.05). For the number of fetal hepatic reticular fibers, there were differences found in RG1 group relative to the CG group (P < 0.05).
2.6. TP, ALB, GLB, GOT, GPT, and AKP in fetal blood Umbilical cord blood was centrifuged at 3500 × g for 15 min, and the serum was stored at −70 ◦ C. Commercial kits to measure total protein (TP, 20110422, Kehua Biological Cor. Shanghai, China), albumin (ALB, 20110622, Kehua Biological Cor. Shanghai, China), alanine transaminase (ALT, 050801, Prodia diagnostics, Germany), aspartate transaminase (AST, 070201, Prodia diagnostics, Germany) and alkaline phosphatase (AKP, 060701, Prodia diagnostics, Germany) were purchased to determine the concentrations of TP, ALB, AST, ALT and AKP in fetal blood using a Hitachi automatic biochemical analyzer (7600-020, Hitachi Ltd., Tokyo, Japan). TP minus ALB was used to determine the globulin (GLB) level.
3.2. T-AOC, GSH-Px, SOD, and MDA in fetal livers Table 4 shows the effects of maternal undernutrition on the concentrations of T-AOC and MDA and the activities of GSH-Px and SOD in fetal livers. The GSHPx activity and MDA concentration in fetal liver in the RG1 group were higher than those of the CG group (P < 0.05); however, the SOD activity and T-AOC concentration were reduced compared with the CG group (P < 0.05). For the RG2 group, although there were no differences in MDA, SOD and GSH-Px relative to the controls (P > 0.05), decreased T-AOC was found as compared with the controls (P < 0.05).
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Table 3 Effects of maternal undernutrition during late pregnancy on the fetal liver weight, chemical components, and the number of collagen fibers and reticular fibers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
BW (g) Liver weight (g) Liver index (% of BW) Fetal liver to brain weight ratio
3978a 100.30a 2.52 1.94a
3573b 85.29b 2.44 1.76a
3111c 72.17c 2.31 1.37b
74 3.71 0.12 0.077
0.0001 0.0004 0.48 0.0012
Chemical components (g) Water Protein Fat Ash
82.35a 12.38a 2.30a 1.21a
67.04b 11.05a 2.29a 1.14a
56.76c 8.81b 1.83b 0.90b
3.54 0.52 0.14 0.072
0.0014 0.0013 0.049 0.025
Chemical components expressed as percentage of liver weight (%) 79.16 Water 12.63 Protein Fat 2.30 1.20 Ash
78.52 12.38 2.54 1.26
78.64 12.22 2.57 1.27
0.30 0.41 0.14 0.058
0.31 0.80 0.38 0.64
Hepatic fibrosis Number of reticular fiber per HPFb Number of collagen fiber per HPF
212ab 814b
291a 1130a
148b 508c
24 25
0.017 0.0001
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 J ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 J ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 J ME kg BW−0.75 d−1 . b HPF=high power field (100×).
Fig. 1. Effect of maternal undernutrition during late pregnancy on fetal liver collagen fibers and reticular fibers. Frames A to C from sections of fetal liver tissues show Masson stain for quantification of collagen fibers; magnification, 200× (the arrow indicates a collagen fiber). Bars, 50 m. Frames D to F show reticulin stain for quantification of reticular fibers; magnification, 400× (the arrow indicates a reticular fiber). Bars = 20 m. Table 4 Effects of maternal undernutrition during late pregnancy on concentrations of T-AOC and MDA, and activities of SOD and GSH-Px in ovine fetal livers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
T-AOC (U/mL)b SOD (U/mL) GSH-PX (mol L−1 ) MDA (nmol/mL)
2.34a 23.60a 24.29b 3.23b
1.30b 22.54ab 24.47b 4.17ab
1.20b 19.33b 34.49a 4.87a
0.13 1.09 2.38 0.42
0.0001 0.043 0.018 0.048
Within a row, means without a common letters (a and b) differ (P < 0.05). a CG = control group, ad libitum, 0.67 J ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 J ME kg BW−0.75 d−1 . b T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.
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Table 5 Effects of maternal undernutrition during late pregnancy on NO, NOs, CHE, and MAO in fetal livers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
NO (umol/g) NOs (U/mg) CHEb (U/mg) MAO (U/mg)
1.86b 0.62b 10.26a 1.52c
2.18ab 0.87a 7.17b 2.32b
2.46a 0.97a 6.86b 2.73a
0.18 0.046 0.38 0.12
0.044 0.0004 0.0001 0.0001
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 MJ ME kg BW−0.75 d−1 . b NO = nitric oxide; NOs = nitric oxide synthase; CHE = cholinesterase; MAO = monoamine oxidase.
3.3. NO, NOs, CHE, and MAO in fetal livers The concentrations of NO, NOs, CHE and MAO in the fetal livers are summarized in Table 5. The concentrations of NOs and MAO in the RG1 and RG2 groups were higher than those of the CG group (P < 0.05), and there was increased NO in RG1 fetuses relative to the controls (P < 0.05); however, the concentrations of CHE in both restricted groups were reduced compared with the CG group (P < 0.05). For MAO, there was a difference between the RG1 and RG2 groups (P < 0.05). 3.4. TP, ALB, GLB, GOT, GPT, and AKP in fetal blood The effects of maternal undernutrition during late pregnancy on the concentrations of TP, ALB, GLB, AST, ALT, and AKP in fetal blood are shown in Table 6. Fetuses with severe IUGR (RG1) exhibited decreased TP, GLB, and ALT relative to the controls (P < 0.05), and their ALB/GLB, AST, and AST/ALT levels were higher than those of the CG group (P < 0.05). For the RG2 group, no difference was observed compared with the CG group (P > 0.05). 4. Discussion Although liver mass and its later function are essentially set in utero during fetal development, it can be ultimately regulated by the intrauterine environment (Hyatt et al., 2008). In sheep, fetal liver growth is sensitive to changes in the materno-fetal nutrient supply, and the magnitude of hepatic adaptation is dependent upon the timing and duration of the nutritional insult (Hyatt et al., 2008). In the present study, the fetal BW and fetal liver weights were severely reduced in both the restricted groups, and the
decreased moisture, protein, fat and ash components in the RG1 fetal livers were found when compared with the controls. As an adaptation to maternal undernutrition, the redistribution of fetal cardiac output preserves the development of brain and nervous system at the expense of the liver and kidneys, which are hypoperfused (Godfrey and Robinson, 1998; Nathanielsz and Hanson, 2003). This adaptation, known as fetal brain-sparing, may account for the liver growth retardation and compromised chemical component deposition. In addition, with the reduction of maternal energy intake, the IUGR fetal liver is smaller relative to the brain. The asymmetric growth indicates that maternal undernutrition selectively down-regulated the liver growth in IUGR fetuses. Malondialdehyde (MDA), as a metabolic product of lipid peroxides (Zhan et al., 2007), is an index of ROS-induced oxidative stress (Wallace, 2005). The data in this study indicate that there was an increasing trend in MDA associated with the extent of maternal feed restriction, and significantly increased MDA was found in the RG1 fetuses. Oxidative stress occurs as a consequence of the imbalance between natural cellular antioxidative defenses and the prooxidant state (Abd Hamid et al., 2011). As an enzyme in antioxidant system, SOD promotes the conversion of an anion superoxide to H2 O2 (Al-Gubory et al., 2010), and GSH-Px prevents free radical damage to phospholipid membranes, enzymes, and other important molecules (Mruk et al., 2002). For the RG1 group, despite the increased GSH-Px, the higher concentration of MDA and reduction of T-AOC in the fetal liver suggest that oxidative stress was induced. The lower SOD activity and higher NO in the RG1 fetal liver might associate with their decreased T-AOC and increased MDA. Nitric oxide operates in a variety of tissues to regulate a diverse range of physiological processes
Table 6 Effects of maternal undernutrition during late pregnancy on TP, ALB, GLB, AST, ALT, and AKP in fetal blood. Items b
TP (g/L) ALB (g/L) GLB (g/L) ALB/GLB AST (IU/L) ALT (IU/L) AST/ALT AKP (IU/L)
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
37.08a 25.28 11.80a 2.15b 27.40b 4.50a 6.11b 275
34.94ab 24.18 10.76ab 2.25ab 27.75b 3.4ab 8.34ab 259
33.87b 23.18 10.22b 2.27a 36.80a 2.8b 13.61a 220
1.04 0.67 0.40 0.032 2.77 0.42 1.85 24
0.039 0.16 0.022 0.026 0.035 0.045 0.030 0.28
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 MJ ME kg BW−0.75 d−1 . b TP = total protein; ALB = albumin; GLB = globulin; AST = aspartate transaminase; ALT = alanine transaminase; AKP = alkaline phosphatase.
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(Dawson and Dawson, 1995), but high levels of NO react with superoxide anions, leading to formation of peroxynitrite radicals (Beckman et al., 1990) that oxidize long-chain fatty acids in cell membranes and result in increases in lipid peroxidation and formation of free radicals (Wang et al., 2002). For the RG2 fetuses, although there were no differences in NO, SOD, GSH-Px, and MDA, decreased TAOC and increased NOs were observed, suggesting a risk of imbalance. Consequently, the antioxidant system of the ovine fetal livers progressively weakened associated with the extent of maternal feed restriction. Monoamine oxidase (MAO) is a family of enzymes that catalyze the oxidation of monoamines (Tipton et al., 2004). Monoamine oxidase is positively correlated with liver fibrosis, and higher MAO in serum or in homogenates always act as an indicator of occurrence for liver fibrosis (Gressner et al., 1982; Chen et al., 2013). Hepatic fibrosis is an important cause of the disruption of normal hepatic architecture and liver dysfunction (Jiao et al., 2009). In the present study, with the reduction of maternal energy intake, the increasing of collagen fibers and reticular fibers in the IUGR fetal liver tissue were observed, and IUGR caused liver fibrosis accompanied by a marked increased MAO in RG1 and RG2 fetal livers. Hepatic fibrosis is a common pathological process resulting from various chronic hepatic injuries and is characterized by an increase of extracellular matrix deposition (Guyton and Hall, 2006). The pathogenesis of liver fibrosis involves a range of causes, but most of them involve the production of free radicals (Jiao et al., 2009), and oxidative stress is a key factor (Chen et al., 2013), which is consistent with the MDA and T-AOC results in this study. Fetal hepatic fibrosis resulting from maternal undernutrition is a worse signal for its liver function and postnatal health. As a remarkable organ with the greatest number of functions, the liver is responsible for coordinating metabolic homeostasis, nutrient processing and detoxification (Hyatt et al., 2008). Fetal livers, the major site of hematopoiesis during embryonic development, acquire additional various metabolic functions at the late gestation or perinatal stage (Haber et al., 1995; Kamiya et al., 1999). Liver function tests are useful in the evaluation of hepatic dysfunction, including tests of ALT and AST, AKP, bilirubin and albumin (Limdi and Hyde, 2003; Ogunkeye and Roluga, 2006). They reflect different functions of the liver, including hepatocellular integrity (transaminases), formation and the subsequent free flow of bile (bilirubin and AKP), and protein synthesis (Limdi and Hyde, 2003). In our study, the fetuses with severe IUGR (RG1) exhibited decreased TP, GLB and ALT, but their ALB/GLB, AST, AST/ALT levels were higher than those of the CG group, which suggested that the IUGR resulting from maternal undernutrition impaired fetal liver function, leading to reduced hepatocellular integrity and poor protein synthesis. However, none of these tests can individually confirm liver dysfunction, and these tests often reveal abnormal results with clinical problems other than liver dysfunction (Weisinger, 2000; Ogunkeye and Roluga, 2006). Cholinesterase (CHE) is synthesized mainly in hepatocytes, and its synthesis is decreased markedly with hepatocyte dysfunction but restored with hepatocyte recovery (Brown et al., 1981; Ogunkeye and Roluga,
2006), which makes cholinesterase a more specific indicator of liver dysfunction than traditional liver function tests (Ogunkeye and Roluga, 2006; Montagnese et al., 2007). As a marker of the overall functional reserve of the liver, cholinesterase may be influenced by nutritional status (Montagnese et al., 2007). In this present study, the concentrations of CHE in both restricted groups were reduced compared with the CG group, which suggests the fetal liver dysfunction was sensitive to maternal undernutrition. The hepatic fibrosis in both restricted groups might be an important cause of their liver dysfunction. In conclusion, the retarded fetal hepatic growth, hepatic fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition during late pregnancy. Conflicts of interest The authors have nothing to disclose and no conflicts of interest to report. Acknowledgments This work was supported by National Natural Science Foundation of China (31260559 and 30800788, Beijing), Inner Mongolia Natural Science Foundation (2013JQ02, Hohhot) and Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (NJYT-12-B09, Hohhot). We gratefully acknowledge Dr. W. Bruce Currie (Emeritus Professor, Cornell University) for help with the manuscript. References Abd Hamid, N.A., Hasrul, M.A., Ruzanna, R.J., Ibrahim, I.A., Baruah, P.S., Mazlan, M., Yusof, Y.A., Ngah, W.Z., 2011. Effect of vitamin E on antioxidant enzymes and DNA damage in rats following eight weeks exercise. Nutr. J. 10, 1–7. Al-Gubory, K.H., Garrel, C., Delatouche, L., Heyman, Y., Chavatte-Palmer, P., 2010. Antioxidant adaptive responses of extraembryonic tissues from cloned and non-cloned bovine conceptuses to oxidative stress during early pregnancy. Reproduction 140, 175–181. Barker, D.J.P., 1997. The long-term outcome of retarded fetal growth. Clin. Obstet. Gynecol. 40, 853–863. Bauer, M., Breier, B., Harding, J., Veldhuis, J., Gluckman, P., 1995. The fetal somatotropic axis during long term maternal undernutrition in sheep: evidence for nutritional regulation in utero. Endocrinology 136, 1250–1257. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87, 1620–1624. Bernstein, I.M., Horbar, J.D., Badger, G.J., Ohlsson, A., Golan, A., 2000. Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restriction. Am. J. Obstet. Gynecol. 182, 198–206. Brown, S.S., Kalow, W., Pilz, W., Whittaker, M., Woronick, C.L., 1981. The plasma cholinesterases: a new perspective. Adv. Clin. Chem. 22, 1–123. Bubb, K.J., Cock, M.L., Black, M.J., Dodic, M., Boon, W.M., Parkington, H.C., Harding, R., Tare, M., 2007. Intrauterine growth restriction delays cardiomyocyte maturation and alters coronary artery function in the fetal sheep. J. Physiol. 578, 871–881. Chen, S., Zou, L., Li, L., Wu, T., 2013. The protective effect of glycyrrhetinic acid on carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of Nrf2. PLoS ONE 8 (1), e53662, http://dx.doi.org/10.1371/journal.pone.0053662. Dawson, T.M., Dawson, V.L., 1995. Nitric oxide: actions and pathological roles. Neuroscientist 1, 7–18.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver Feng Gao a,∗ , Yingchun Liu b , Lingyao Li a , Ming Li a , Chongzhi Zhang a , Changjin Ao a , Xianzhi Hou a a b
College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 25 April 2014 Accepted 26 April 2014 Available online xxx
Keywords: Fetal growth restriction Maternal undernutrition Hepatic development Anti-oxidation capability Liver function
a b s t r a c t This study investigated the effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Eighteen ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.175 MJ ME kg BW−0.75 d−1 , n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW−0.75 d−1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 , n = 6). Fetuses were recovered at slaughter on d 140. Fetuses in the RG1 group exhibited decreased (P < 0.05) liver weight, total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), cholinesterase (CHE), total protein (TP), globulin (GLB), and alanine transaminase (ALT). In addition, intermediate changes were found in the RG2 fetuses, including decreased liver weight, T-AOC and CHE (P < 0.05). In contrast, increases in fetal hepatic collagen fibers and reticular fibers, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOs), monoamine oxidase (MAO), albumin (ALB)/GLB, aspartate transaminase (AST), and AST/ALT were found in the RG1 fetuses (P < 0.05). The RG2 fetuses had increased fetal hepatic collagen fibers, NOs and MAO (P < 0.05) relative to the control fetuses. These results indicate that impaired fetal hepatic growth, fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Intrauterine growth restriction (IUGR), often resulting from maternal undernutrition during pregnancy (Robinson et al., 1999; McMillen et al., 2001), is a significant cause of fetal and neonatal mortality and morbidity (Bernstein et al., 2000; Wu et al., 2006). Considerable epidemiological data indicate that the growth of the heart (Han et al., 2004; Bubb et al., 2007), lungs (Lipsett et al., 2006), and pancreas
∗ Corresponding author at: College of Animal Science, Inner Mongolia Agricultural University, No. 306# Zhao Wu Da Street, Hohhot 010018, Inner Mongolia, China. Tel.: +86 471 4309175; fax: +86 471 4301530. E-mail address: [email protected] (F. Gao).
(Dumortier et al., 2007) in IUGR fetuses are retarded, and these organs experience structural changes and impaired function that strongly predispose the animals to the development of cardiovascular disease (Barker, 1997), respiratory illness (Maritz et al., 2004), diabetes and insulin resistance (Vuguin et al., 2004; Green et al., 2010) in later life. As the largest of the body’s organs with the greatest number of functions, the developing fetal liver is sensitive to damage from both internal and external sources, including teratogens, infection and nutritional deficiencies (Hyatt et al., 2008). The impaired growth of fetal livers caused by maternal undernutrition has been observed (Bauer et al., 1995; Osgerby et al., 2002; Hyatt et al., 2002; Gao et al., 2009), and altered gene expression of the hepatic PRLGH-IGF axis has been detected in prenatal and postnatal
http://dx.doi.org/10.1016/j.anireprosci.2014.04.012 0378-4320/© 2014 Elsevier B.V. All rights reserved.
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animals (Bauer et al., 1995; Hyatt et al., 2002, 2004, 2007). Although these data indicate that exposure to an adverse nutritional environment in pregnancy has a long-term impact on fetal or neonatal liver growth, additional studies are needed to ascertain whether reduced liver mass and the expression of growth factors in IUGR fetal sheep affect liver function and, indeed, whether such responses contribute to the developmental programming of poor health in later life (Hyatt et al., 2008). Therefore, the objective of the present study was to test the hypothesis that maternal undernutrition during late pregnancy impairs hepatic growth and is associated with liver dysfunction in IUGR ovine fetuses. 2. Materials and methods
Table 1 Composition of grass hay and the leftover hay during restriction period.
ME (MJ/Kg)a DM (%) CP (%) EE (%) NDF (%) ADF (%) ASH (%) Ca (%) P (%)
Grass hay
Leftover hay
8.90 88.42 10.09 4.34 71.98 35.82 4.67 0.57 0.09
– 91.99 9.27 2.72 71.19 36.60 4.39 0.68 0.08
a ME, metabolizable energy; DM, dry matter; CP, crude protein; NDF, neutraldetergent fiber; ADF, acid detergent fiber, EE = ether extract; Ca = calcium; P = phosphorus.
2.1. Animals and treatments All experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals in China (The State Science and Technology Commission of China, 1988). This study is a companion study, and the details of animals, experimental design and detailed procedures have been presented previously (Gao et al., 2013). Briefly, when the maternal nutrition density during late pregnancy is lower than the ensured “threshold” level (0.33 MJ ME kg BW−0.75 d−1 ), it may lead to Mongolian ovine fetal pathological changes and the ability of compensatory growth of postnatal offspring is suppressed or even lost (Gao, 2006; Qi, 2007; Gao et al., 2007, 2008, 2009). According to the previous results, three groups comprising eighteen Mongolian ewes carrying singletons were allocated at d 90 of pregnancy: a severely restricted group (Restricted Group 1: RG1, 0.175 MJ ME kg BW−0.75 d−1 , n = 6), a restricted group at the “threshold” level (Restricted Group 2: RG2, 0.33 MJ ME kg BW−0.75 d−1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 , n = 6). Second or third parity ewes were mated at synchronized estrus and had similar live weights (mean 52.82 ± 2.67 kg) during the d 90 of pregnancy. Pregnancies were confirmed by ultrasound scanning at approximately d 50 of gestation (Medison-SA-600, Shanghai, China). All animals were housed in individual pens, and chopped hay (mainly Leymus chinensis) was supplied ad libitum until d 90 of gestation. Based on the fact that the fetus is considered to achieve 80% to 85% of its final birth weight during the last two months of gestation (Robinson et al., 1999; Symonds et al., 2001), maternal undernutrition was imposed from d 90 to d 140 of pregnancy. At the beginning of restriction, the ME and chemical composition in the hay were measured (Table 1), and then the daily intake of the hay offered in RG1 and RG2 was calculated by the ewe body weight, nutrition value of hay, and the designed energy plane in the restricted groups. Restricted ewes were fed at 08:30 and 16:00 h each day, and the amount of feed offered was constant throughout the restriction period (Table 2). The ewes in the Control Group were offered feed at 08:30, 11:00 and 16:00 h daily (the feed refusals were approximately 10% of the total amount offered). The animals had free access to water and mineral mixture blocks (containing per kilogram: Ca, 15 g; P, 11.5 g; Mg as MgSO4 ·H2 O, 1 g; Fe as FeSO4 ·7H2 O, 500 mg;
Cu as CuSO4 ·5H2 O, 250 mg; Zn as ZnSO4 , 175 mg; Mn as MnSO4 , 100 mg; Co as CoCl2 ·6H2 O, 20 mg; I as KI, 40 mg; Se as Na2 SeO3 ·5H2 O, 1.5 mg; Yuantongweiye Co., Ltd., Inner Mongolian, China). All feed refusals were collected daily before feeding at 08:30, weighed and sub-sampled for chemical analysis.
2.2. Slaughtering procedures All fetuses were removed at d 140 of gestation. The umbilical cord blood was collected, and the fetal BW and liver weights were recorded. Some of the liver tissue was snap-frozen in liquid nitrogen and stored at −80 ◦ C. Tissue samples (approximately 1.0 cm3 ) were harvested from the large lobes of the liver. After rinsing with phosphatebuffered saline (PBS, pH 7.4), portions of the fetal liver were immediately placed in paraformaldehyde fixative solution (0.1 mol L−1 7.4). After fixation for at least 2 days, the tissues were dehydrated and paraffin-embedded, sectioned at 4–6 m and stained with commercial kits of reticulin stain (D032, NJJCBIO, Nanjing, China) and Masson stain (D026, NJJCBIO, Nanjing, China) for microscopic examination. Thereafter, five images were obtained from each section. Each specimen was viewed under a standard microscope, and the total number of collagen fibers and reticular fibers were counted in 10 random high power fields (HPF, magnification 100×) by three observers who were blinded to the study groups and results. Data were expressed as the number of collagen fibers per field and reticular fibers per field.
2.3. Liver chemical components analyses Fetal liver moisture was determined by freeze-drying to a constant weight (Christ, Alpha 1-4 lsc, German), and sample components were analyzed for chemical fat, protein and ash. The crude protein content was determined using the Kjeldahl method, and the values were converted to protein using the factor 6.25. The chemical fat content was determined as the difference in DM before and after extraction using ether. The ash content of the fetal liver was the residue left after ashing at 550 ◦ C in a muffle furnace.
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Table 2 Planes of maternal nutrition in different groups during late pregnancy. Nutrition levels
CG (ad libitum)a
RG2
RG1
Mean daily grass intake (g/d)b Mean daily crude protein intake (g/d) Daily metabolizable energy intakec (MJ ME kg BW−0.75 d−1 )
1689.05 170.43 0.670
842.49 85.01 0.330
440.18 44.41 0.175
a b c
CG = control group; RG2 = restricted group 2; RG1 = restricted group 1. Mean daily grass intake and crude protein intake are represented on a natural basis. Daily etabolizable energy intake is represented on a dry matter basis.
2.4. T-AOC, GSH-Px, SOD, and MDA in fetal livers
2.7. Statistical analysis
Approximately 0.5 g of the fetal liver was rinsed and homogenized in 0.85% chilled normal saline to obtain a 10% liver homogenate for the measurement of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. The activities of GSH-Px (A005) and SOD (A001-1), and MDA (A003-1) content were determined spectrophotometrically using commercial kits (NJJCBIO, Nanjing, China) according to the procedures of Paglia and Valentine (1967), Panckenko et al. (1975), and Placer et al. (1966), respectively. The total antioxidant capacity was determined using a spectrometric commercial kit (A015, NJJCBIO, Nanjing, China). In the reaction mixture, ferric ion was reduced by antioxidant reducing agents, and the blue complex Fe2+ -TPTZ (2,4,6tri (2-pyridyl)-s-triazine) was produced; absorbance was measured at 520 nm. One unit of T-AOC was defined as the amount that increased the absorbance by 0.01 at 37 ◦ C and was expressed as units per milliliter in the tissue homogenate.
All data were analyzed by using the ANOVA procedure as implemented in SAS software (the SAS Institute Inc, 2001). The model was as follows: Yi = u + Mi + ei , where u is the overall mean. Mi is the fixed effect of the nutrition treatments (i = 1–3), and ei is the random residual error. Duncan’s test was used to identify significant differences between mean values. Significance was declared at P ≤ 0.05.
2.5. NO, NOs, CHE and MAO in fetal livers A 10% liver homogenate was used to analyze the concentrations of the nitric oxide (NO, A013-1), nitric oxide synthase (NOs, A014-2), cholinesterase (CHE, 20120914), and monoamine oxidase (MAO, 20120914) in fetal livers according to the procedures of commercial kits (NJJCBIO, Nanjing, China). Each measurement was performed in duplicate.
3. Results 3.1. Fetal liver weights, structures and chemical component The effects of maternal undernutrition on the fetal liver weights, structures and chemical components are presented in Table 3 and Fig. 1. Fetal BW (P = 0.0001), liver weights (P = 0.0004) and fetal liver water content (P = 0.0014) in the restricted groups were reduced compared with the CG group, and there were differences in the fetal BW, liver weights and water content between the RG1 and RG2 groups (P < 0.05). In addition, the decreased protein, fat and ash components in fetal livers and a reduced fetal liver to brain weight ratio were found in RG1 as compared with the controls (P < 0.05). With the reduction of maternal energy intake, the number of fetal hepatic collagen fibers and reticular fibers per HPF in the RG1 and RG2 groups was increased, and there were differences in the number of collagen fibers among the RG1, RG2 and CG groups (P < 0.05). For the number of fetal hepatic reticular fibers, there were differences found in RG1 group relative to the CG group (P < 0.05).
2.6. TP, ALB, GLB, GOT, GPT, and AKP in fetal blood Umbilical cord blood was centrifuged at 3500 × g for 15 min, and the serum was stored at −70 ◦ C. Commercial kits to measure total protein (TP, 20110422, Kehua Biological Cor. Shanghai, China), albumin (ALB, 20110622, Kehua Biological Cor. Shanghai, China), alanine transaminase (ALT, 050801, Prodia diagnostics, Germany), aspartate transaminase (AST, 070201, Prodia diagnostics, Germany) and alkaline phosphatase (AKP, 060701, Prodia diagnostics, Germany) were purchased to determine the concentrations of TP, ALB, AST, ALT and AKP in fetal blood using a Hitachi automatic biochemical analyzer (7600-020, Hitachi Ltd., Tokyo, Japan). TP minus ALB was used to determine the globulin (GLB) level.
3.2. T-AOC, GSH-Px, SOD, and MDA in fetal livers Table 4 shows the effects of maternal undernutrition on the concentrations of T-AOC and MDA and the activities of GSH-Px and SOD in fetal livers. The GSHPx activity and MDA concentration in fetal liver in the RG1 group were higher than those of the CG group (P < 0.05); however, the SOD activity and T-AOC concentration were reduced compared with the CG group (P < 0.05). For the RG2 group, although there were no differences in MDA, SOD and GSH-Px relative to the controls (P > 0.05), decreased T-AOC was found as compared with the controls (P < 0.05).
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Table 3 Effects of maternal undernutrition during late pregnancy on the fetal liver weight, chemical components, and the number of collagen fibers and reticular fibers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
BW (g) Liver weight (g) Liver index (% of BW) Fetal liver to brain weight ratio
3978a 100.30a 2.52 1.94a
3573b 85.29b 2.44 1.76a
3111c 72.17c 2.31 1.37b
74 3.71 0.12 0.077
0.0001 0.0004 0.48 0.0012
Chemical components (g) Water Protein Fat Ash
82.35a 12.38a 2.30a 1.21a
67.04b 11.05a 2.29a 1.14a
56.76c 8.81b 1.83b 0.90b
3.54 0.52 0.14 0.072
0.0014 0.0013 0.049 0.025
Chemical components expressed as percentage of liver weight (%) 79.16 Water 12.63 Protein Fat 2.30 1.20 Ash
78.52 12.38 2.54 1.26
78.64 12.22 2.57 1.27
0.30 0.41 0.14 0.058
0.31 0.80 0.38 0.64
Hepatic fibrosis Number of reticular fiber per HPFb Number of collagen fiber per HPF
212ab 814b
291a 1130a
148b 508c
24 25
0.017 0.0001
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 J ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 J ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 J ME kg BW−0.75 d−1 . b HPF=high power field (100×).
Fig. 1. Effect of maternal undernutrition during late pregnancy on fetal liver collagen fibers and reticular fibers. Frames A to C from sections of fetal liver tissues show Masson stain for quantification of collagen fibers; magnification, 200× (the arrow indicates a collagen fiber). Bars, 50 m. Frames D to F show reticulin stain for quantification of reticular fibers; magnification, 400× (the arrow indicates a reticular fiber). Bars = 20 m. Table 4 Effects of maternal undernutrition during late pregnancy on concentrations of T-AOC and MDA, and activities of SOD and GSH-Px in ovine fetal livers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
T-AOC (U/mL)b SOD (U/mL) GSH-PX (mol L−1 ) MDA (nmol/mL)
2.34a 23.60a 24.29b 3.23b
1.30b 22.54ab 24.47b 4.17ab
1.20b 19.33b 34.49a 4.87a
0.13 1.09 2.38 0.42
0.0001 0.043 0.018 0.048
Within a row, means without a common letters (a and b) differ (P < 0.05). a CG = control group, ad libitum, 0.67 J ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 J ME kg BW−0.75 d−1 . b T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.
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Table 5 Effects of maternal undernutrition during late pregnancy on NO, NOs, CHE, and MAO in fetal livers. Items
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
NO (umol/g) NOs (U/mg) CHEb (U/mg) MAO (U/mg)
1.86b 0.62b 10.26a 1.52c
2.18ab 0.87a 7.17b 2.32b
2.46a 0.97a 6.86b 2.73a
0.18 0.046 0.38 0.12
0.044 0.0004 0.0001 0.0001
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 MJ ME kg BW−0.75 d−1 . b NO = nitric oxide; NOs = nitric oxide synthase; CHE = cholinesterase; MAO = monoamine oxidase.
3.3. NO, NOs, CHE, and MAO in fetal livers The concentrations of NO, NOs, CHE and MAO in the fetal livers are summarized in Table 5. The concentrations of NOs and MAO in the RG1 and RG2 groups were higher than those of the CG group (P < 0.05), and there was increased NO in RG1 fetuses relative to the controls (P < 0.05); however, the concentrations of CHE in both restricted groups were reduced compared with the CG group (P < 0.05). For MAO, there was a difference between the RG1 and RG2 groups (P < 0.05). 3.4. TP, ALB, GLB, GOT, GPT, and AKP in fetal blood The effects of maternal undernutrition during late pregnancy on the concentrations of TP, ALB, GLB, AST, ALT, and AKP in fetal blood are shown in Table 6. Fetuses with severe IUGR (RG1) exhibited decreased TP, GLB, and ALT relative to the controls (P < 0.05), and their ALB/GLB, AST, and AST/ALT levels were higher than those of the CG group (P < 0.05). For the RG2 group, no difference was observed compared with the CG group (P > 0.05). 4. Discussion Although liver mass and its later function are essentially set in utero during fetal development, it can be ultimately regulated by the intrauterine environment (Hyatt et al., 2008). In sheep, fetal liver growth is sensitive to changes in the materno-fetal nutrient supply, and the magnitude of hepatic adaptation is dependent upon the timing and duration of the nutritional insult (Hyatt et al., 2008). In the present study, the fetal BW and fetal liver weights were severely reduced in both the restricted groups, and the
decreased moisture, protein, fat and ash components in the RG1 fetal livers were found when compared with the controls. As an adaptation to maternal undernutrition, the redistribution of fetal cardiac output preserves the development of brain and nervous system at the expense of the liver and kidneys, which are hypoperfused (Godfrey and Robinson, 1998; Nathanielsz and Hanson, 2003). This adaptation, known as fetal brain-sparing, may account for the liver growth retardation and compromised chemical component deposition. In addition, with the reduction of maternal energy intake, the IUGR fetal liver is smaller relative to the brain. The asymmetric growth indicates that maternal undernutrition selectively down-regulated the liver growth in IUGR fetuses. Malondialdehyde (MDA), as a metabolic product of lipid peroxides (Zhan et al., 2007), is an index of ROS-induced oxidative stress (Wallace, 2005). The data in this study indicate that there was an increasing trend in MDA associated with the extent of maternal feed restriction, and significantly increased MDA was found in the RG1 fetuses. Oxidative stress occurs as a consequence of the imbalance between natural cellular antioxidative defenses and the prooxidant state (Abd Hamid et al., 2011). As an enzyme in antioxidant system, SOD promotes the conversion of an anion superoxide to H2 O2 (Al-Gubory et al., 2010), and GSH-Px prevents free radical damage to phospholipid membranes, enzymes, and other important molecules (Mruk et al., 2002). For the RG1 group, despite the increased GSH-Px, the higher concentration of MDA and reduction of T-AOC in the fetal liver suggest that oxidative stress was induced. The lower SOD activity and higher NO in the RG1 fetal liver might associate with their decreased T-AOC and increased MDA. Nitric oxide operates in a variety of tissues to regulate a diverse range of physiological processes
Table 6 Effects of maternal undernutrition during late pregnancy on TP, ALB, GLB, AST, ALT, and AKP in fetal blood. Items b
TP (g/L) ALB (g/L) GLB (g/L) ALB/GLB AST (IU/L) ALT (IU/L) AST/ALT AKP (IU/L)
CGa (n = 6)
RG2 (n = 6)
RG1 (n = 6)
SEM
P-value
37.08a 25.28 11.80a 2.15b 27.40b 4.50a 6.11b 275
34.94ab 24.18 10.76ab 2.25ab 27.75b 3.4ab 8.34ab 259
33.87b 23.18 10.22b 2.27a 36.80a 2.8b 13.61a 220
1.04 0.67 0.40 0.032 2.77 0.42 1.85 24
0.039 0.16 0.022 0.026 0.035 0.045 0.030 0.28
Within a row, means without a common letters (a–c) differ (P < 0.05). a CG = control group, ad libitum, 0.67 MJ ME kg BW−0.75 d−1 ; RG2 = restricted group 2, 0.33 MJ ME kg BW−0.75 d−1 ; RG1 = restricted group 1, 0.175 MJ ME kg BW−0.75 d−1 . b TP = total protein; ALB = albumin; GLB = globulin; AST = aspartate transaminase; ALT = alanine transaminase; AKP = alkaline phosphatase.
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(Dawson and Dawson, 1995), but high levels of NO react with superoxide anions, leading to formation of peroxynitrite radicals (Beckman et al., 1990) that oxidize long-chain fatty acids in cell membranes and result in increases in lipid peroxidation and formation of free radicals (Wang et al., 2002). For the RG2 fetuses, although there were no differences in NO, SOD, GSH-Px, and MDA, decreased TAOC and increased NOs were observed, suggesting a risk of imbalance. Consequently, the antioxidant system of the ovine fetal livers progressively weakened associated with the extent of maternal feed restriction. Monoamine oxidase (MAO) is a family of enzymes that catalyze the oxidation of monoamines (Tipton et al., 2004). Monoamine oxidase is positively correlated with liver fibrosis, and higher MAO in serum or in homogenates always act as an indicator of occurrence for liver fibrosis (Gressner et al., 1982; Chen et al., 2013). Hepatic fibrosis is an important cause of the disruption of normal hepatic architecture and liver dysfunction (Jiao et al., 2009). In the present study, with the reduction of maternal energy intake, the increasing of collagen fibers and reticular fibers in the IUGR fetal liver tissue were observed, and IUGR caused liver fibrosis accompanied by a marked increased MAO in RG1 and RG2 fetal livers. Hepatic fibrosis is a common pathological process resulting from various chronic hepatic injuries and is characterized by an increase of extracellular matrix deposition (Guyton and Hall, 2006). The pathogenesis of liver fibrosis involves a range of causes, but most of them involve the production of free radicals (Jiao et al., 2009), and oxidative stress is a key factor (Chen et al., 2013), which is consistent with the MDA and T-AOC results in this study. Fetal hepatic fibrosis resulting from maternal undernutrition is a worse signal for its liver function and postnatal health. As a remarkable organ with the greatest number of functions, the liver is responsible for coordinating metabolic homeostasis, nutrient processing and detoxification (Hyatt et al., 2008). Fetal livers, the major site of hematopoiesis during embryonic development, acquire additional various metabolic functions at the late gestation or perinatal stage (Haber et al., 1995; Kamiya et al., 1999). Liver function tests are useful in the evaluation of hepatic dysfunction, including tests of ALT and AST, AKP, bilirubin and albumin (Limdi and Hyde, 2003; Ogunkeye and Roluga, 2006). They reflect different functions of the liver, including hepatocellular integrity (transaminases), formation and the subsequent free flow of bile (bilirubin and AKP), and protein synthesis (Limdi and Hyde, 2003). In our study, the fetuses with severe IUGR (RG1) exhibited decreased TP, GLB and ALT, but their ALB/GLB, AST, AST/ALT levels were higher than those of the CG group, which suggested that the IUGR resulting from maternal undernutrition impaired fetal liver function, leading to reduced hepatocellular integrity and poor protein synthesis. However, none of these tests can individually confirm liver dysfunction, and these tests often reveal abnormal results with clinical problems other than liver dysfunction (Weisinger, 2000; Ogunkeye and Roluga, 2006). Cholinesterase (CHE) is synthesized mainly in hepatocytes, and its synthesis is decreased markedly with hepatocyte dysfunction but restored with hepatocyte recovery (Brown et al., 1981; Ogunkeye and Roluga,
2006), which makes cholinesterase a more specific indicator of liver dysfunction than traditional liver function tests (Ogunkeye and Roluga, 2006; Montagnese et al., 2007). As a marker of the overall functional reserve of the liver, cholinesterase may be influenced by nutritional status (Montagnese et al., 2007). In this present study, the concentrations of CHE in both restricted groups were reduced compared with the CG group, which suggests the fetal liver dysfunction was sensitive to maternal undernutrition. The hepatic fibrosis in both restricted groups might be an important cause of their liver dysfunction. In conclusion, the retarded fetal hepatic growth, hepatic fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition during late pregnancy. Conflicts of interest The authors have nothing to disclose and no conflicts of interest to report. Acknowledgments This work was supported by National Natural Science Foundation of China (31260559 and 30800788, Beijing), Inner Mongolia Natural Science Foundation (2013JQ02, Hohhot) and Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (NJYT-12-B09, Hohhot). We gratefully acknowledge Dr. W. Bruce Currie (Emeritus Professor, Cornell University) for help with the manuscript. References Abd Hamid, N.A., Hasrul, M.A., Ruzanna, R.J., Ibrahim, I.A., Baruah, P.S., Mazlan, M., Yusof, Y.A., Ngah, W.Z., 2011. Effect of vitamin E on antioxidant enzymes and DNA damage in rats following eight weeks exercise. Nutr. J. 10, 1–7. Al-Gubory, K.H., Garrel, C., Delatouche, L., Heyman, Y., Chavatte-Palmer, P., 2010. Antioxidant adaptive responses of extraembryonic tissues from cloned and non-cloned bovine conceptuses to oxidative stress during early pregnancy. Reproduction 140, 175–181. Barker, D.J.P., 1997. The long-term outcome of retarded fetal growth. Clin. Obstet. Gynecol. 40, 853–863. Bauer, M., Breier, B., Harding, J., Veldhuis, J., Gluckman, P., 1995. The fetal somatotropic axis during long term maternal undernutrition in sheep: evidence for nutritional regulation in utero. Endocrinology 136, 1250–1257. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87, 1620–1624. Bernstein, I.M., Horbar, J.D., Badger, G.J., Ohlsson, A., Golan, A., 2000. Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restriction. Am. J. Obstet. Gynecol. 182, 198–206. Brown, S.S., Kalow, W., Pilz, W., Whittaker, M., Woronick, C.L., 1981. The plasma cholinesterases: a new perspective. Adv. Clin. Chem. 22, 1–123. Bubb, K.J., Cock, M.L., Black, M.J., Dodic, M., Boon, W.M., Parkington, H.C., Harding, R., Tare, M., 2007. Intrauterine growth restriction delays cardiomyocyte maturation and alters coronary artery function in the fetal sheep. J. Physiol. 578, 871–881. Chen, S., Zou, L., Li, L., Wu, T., 2013. The protective effect of glycyrrhetinic acid on carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of Nrf2. PLoS ONE 8 (1), e53662, http://dx.doi.org/10.1371/journal.pone.0053662. Dawson, T.M., Dawson, V.L., 1995. Nitric oxide: actions and pathological roles. Neuroscientist 1, 7–18.
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