Effect of maternal low protein diet during pregnancy on the fetal liver of rats

Annals of Anatomy 195 (2013) 68–76

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Research article

Effect of maternal low protein diet during pregnancy on the fetal liver of rats Wafaa S. Ramadan a,b,∗ , Ilham Alshiraihi c , Saleh Al-karim c a

Department of Anatomy, Faculty of Medicine, KAU, Saudi Arabia Department of Anatomy, Faculty of Medicine, Ain Shams University, Egypt c Department of Biological Sciences, Faculty of Science, KAU, Saudi Arabia b

a r t i c l e

i n f o

Article history: Received 15 November 2011 Received in revised form 13 May 2012 Accepted 14 May 2012 Keywords: Maternal low Fetal hepatocyte TUNEL Ki-67 Ultrastructure Glycogen

a b s t r a c t Maternal protein restriction plays a critical role in the developmental programming of later disease susceptibility of the fetus. Developmental insults could exert permanent effects on health through alteration of tissue morphology. As the liver has the greatest number of functions among other body organs, this study aimed at evaluating the effects of maternal dietary protein insufficiency on the structure and the proliferative capacity of the liver in rat fetuses. Morphometric histological studies and biochemical analysis were performed. Twenty adult Albino female Wistar rats were divided into two groups after confirmation of pregnancy. Group I (ST), serving as control, was fed a standard diet (20% protein) and group II (LP) a low protein diet (5% protein). Fetuses were extracted on the day 21.5 of pregnancy. Group II morphometric results revealed a significant decrease in the mothers’ weight gain, number and weight of fetuses and weight of fetal livers, but there was also an increase in the mean area of hepatocytes. Histological results showed apoptosis, vacuolization of the hepatocytes, increased positivity of the Oil Red O stained fat droplets and the PAS-positive stained glycogen granules. Liver TUNEL showed increased apoptotic nuclei. Ki-67 immunostaining showed decreased proliferation of the hepatocytes. Ultrastructurally, the nucleus showed peripheral masses of heterochromatin besides irregular nuclear and cell membranes. Mitochondria varied in shape with loss of cristea. Biochemically, there was a significant decrease in the protein concentration and a significant increase in the glycogen concentration in livers of group II. It thus appears that the maternal metabolic condition not only reduced fetal growth in response to protein restriction, but also altered the structure of the liver. © 2012 Elsevier GmbH. All rights reserved.

1. Introduction Intrinsic or extrinsic utero perturbations can potentially affect the growth and development of the fetus (Cheung et al., 2004). In severe cases, it may lead to intrauterine growth restriction (IUGR) and failure to thrive postnatally (Hales and Barker, 2001). The adaptive changes by the embryo or fetus to an altered intrauterine environment may improve the immediate chances of survival but be detrimental to subsequent postnatal life (Gallagher et al., 2005). It also may play a critical role in the developmental programming of later disease susceptibility (Ergaz et al., 2005). Programming is the consequence of the innate capacity of developing tissues to adapt to the conditions that prevail during early life. For almost all cell types in all organs, this is an ability that is present for only a short period before the time of birth (Langley-Evans, 2006). The effects of

∗ Corresponding author at: Department of Anatomy, Faculty of Medicine, KAU, Saudi Arabia. Tel.: +966 507951993. E-mail address: [email protected] (W.S. Ramadan). 0940-9602/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.aanat.2012.05.006

programming may pass across generations by mechanisms that do not necessarily involve changes in the genes (Nijland et al., 2008). Maternal malnutrition prior to and during pregnancy manifested by low body weight, short stature and inadequate energy intake during pregnancy are considered major determinants in developing countries where the economic burden is high (Kalhan et al., 2009). Restricted maternal low protein diet during pregnancy and or lactation predisposed the offspring to insulin resistance later in life (Zambrano et al., 2005). In addition to an age-dependent loss of glucose tolerance (Petry et al., 2001), maternal protein restriction in the rat has been shown to be associated with hypertension (Langley-Evans et al., 1999). But the mechanisms underlying reduced fetal growth in response to maternal protein restriction are not well established (Rosario et al., 2011). The simplest process through which developmental insults could exert permanent effects on physiology, metabolism and health is through alteration of tissue morphology. Changes to the numbers of cells or the type of cells present within a tissue could have profound effects on organ function (Langley-Evans, 2006). As the liver has the greatest number of functions among the body organs, it is of great interest to study the detailed account of changes taking place

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in the liver when the protein concentration in the maternal diet is a variable factor.

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diet given during the whole period of pregnancy before dissection on the day 21.5. All procedures were approved by the Animal Experimentation Ethics Committee of King Abdulaziz University.

1.1. Aim of work The present work aimed to evaluate the effects of dietary protein insufficiency during pregnancy on the structure of the liver in rat fetuses. Morphometric and histological studies in addition to biochemical analysis of the glycogen and protein content in the fetal liver will be assessed. Beyond this the proliferative activity of the hepatocytes will be traced using Ki-67 diagnostic perspectives. 2. Materials and methods 2.1. Animals Forty adult Albino Wistar rats (20 male and 20 female rats) weighing between 180 and 200 g were purchased from the animal house of King Fahd Medical Research Center (King Abdulaziz University). The animals were housed in environmentally controlled cages (25◦ C, 12-h light/12-h dark cycle). Tap water and experimental diet was supplied ad libitum. 2.2. Diet Each of the standard and low protein powdered semisynthetic diets consisted of: Standard diet: 68% starch, 4% cellulose, 5% lipid (corn oil) and 20% protein (casein) (g/100 g). Low protein diet: 78% starch, 4% cellulose, 5% lipid (corn oil) and 5% protein (casein) (g/100 g). Both diets contained 2 g/100 g yeast, salt 3.5 g/100 g, and vitamin mixture 2.2 g/100 g (Picarel-Blanchot et al., 1995). Both diets were isocaloric because the protein deficiency in the LP diet was compensated by the addition of carbohydrates. Casein, starch, cellulose and vitamin mixture were purchased from Sigma (USA) brand products through Sigma–Aldrich, Inc. Corn oil, yeast and salt were purchased from local market. 2.3. Biochemical assay kits The protein assay kit was purchased from (Sigma, USA) and the glycogen assay kit from (Biovision, USA). 2.4. Liver samples On the early morning of day 21.5 of pregnancy, rats from both groups (fasted overnight) were anesthetized with pentobarbital sodium for 20 min and dissected via abdominal incision. The uterus was opened longitudinally to extract the fetuses by separating the placenta. The fetuses were counted and weighed. The fetal livers were removed and weighed after a midline incision (Bertin et al., 2002; El-Khattabi et al., 2003). They were prepared for histological studies and biochemical analysis. 2.5. Experimental protocol The animals were allowed to mate together for 1 night by placing one female with one male rat in a cage. The next morning, the presence of sperm in the vaginal smear was confirmed and this was taken as day 0.5 of pregnancy. Midnight was considered the time of mating. The 20 pregnant females were randomly transferred to individual cages and were divided into 2 groups according to the

Group I (ST) (10 pregnant females) this group served as control and was fed on the standard diet. Group II (LP) (10 pregnant females) were fed on low protein diet. 2.6. Morphometric study Pregnant mothers were weighed weekly using a weighing balance for animals (readability 0.01 g). The fetuses were counted and weighed after their removal from the uterus. The wet weights (g) of fetal livers were rapidly determined after their removal from fetuses (El-Khattabi et al., 2003). Images of tissue sections stained with Hematoxylin and Eosin were analyzed using Olysia BioReport software (Olympus-Japan) while those of the immunostained sections were analyzed using Image Pro Plus software Version 6.0 (Media Cybernetics, Inc., USA) (Zaitoun et al., 2005). The total area of the hepatocytes, cytoplasm and nuclei were determined (␮m2 ). At least ten cells in five random fields were analyzed. 2.7. Histological study 2.7.1. Light microscopic study 2.7.1.1. Part of the liver samples were fixed in neutral buffered formalin, dehydrated and then embedded in paraffin wax. Paraffin blocks were cut into sections (5–8 ␮m) using a microtome (Thermo Shandon, UK). The serial sections were mounted on glass slides, hydrated and stained with Hematoxylin and Eosin stain (H&E) (Drury and Wallington, 1980). 2.7.1.2. For staining glycogen periodic acid-Schiff stain (PAS) was used. Sections of the previously prepared paraffin blocks were oxidized for 5 min with aqueous periodic acid, washed, rinsed and then placed for 20 min in Schiff’s reagent (Schiff, 1866). 2.7.1.3. Oil Red O stain was applied for staining of lipids. Some frozen liver samples were cut using cryostat to sections (5 ␮m) at −10 ◦ C to −30 ◦ C (TISSUE TEK, USA). The sections were rinsed in 60% triethyl phosphate, immersed for 10 min in Oil Red O solution, washed and rinsed. The nuclei were stained blue using Hematoxylin as counter stain (Lillie and Ashburn, 1943). 2.7.1.4. Immunostaining. 2.7.1.4.1. Ki-67. Four-␮m-thick formalin-fixed paraffin waxembedded sections from the fetal liver of the two groups dewaxed and rehydrated then incubated with hydrogen peroxide (2.4 ml 30%) in methanol (400 ml) to block endogenous peroxidases. Antigen retrieval was performed by microwaving in sodium citrate. Liver sections were treated with an avidin/biotin kit (DAKO, Cambridgeshire, UK; X0590), blocked in serum rabbit serum diluted 1/25 in PBS (DAKO; Catalog no. X0902) for 15 min and then incubated in the primary antibody Ki-67/MIB 5 (rabbit polyclonal) at dilution 1/200 for 35 min. A biotinylated secondary antibody Swine anti-rabbit of dilution 1/500 (Novocastra, Newcastle, UK NCL-Ki67p) was then applied for 35 min. A layer of streptavidin–horseradish peroxidase (DAKO; P0397) diluted to 1/500 in PBS for 35 min was applied, followed by PBS wash and a 2-min incubation in 3,3 -diaminobenzidine (0.005 g in 10 ml PBS). Sections without the primary antibody were used as negative controls. All sections were counterstained with hematoxylin and mounted (Vig et al., 2006). All hepatocytes with nuclear staining of any intensity were defined as positive.

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2.7.1.4.2. TUNEL technique. Apoptotic nuclei in tissue sections were identified with in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) – biotin nickend labeling (TUNEL) technique according to the kit manufacturer’s protocol (In situ Apoptosis detection Kit, Catalog number 481530-k, Model number 039M) (Trevigen, Gaithersburg, MD). Sections were deparaffinized and rinsed in distilled water. After proteinase K treatment and removal of endogenous peroxidase activity with hydrogen peroxide, sections were incubated at 37 ◦ C with TdTdeoxynucleoside triphosphate (dNTP)-manganese reaction buffer. After 1 h, the reaction was stopped and the slides were washed in PBS. Nuclear labeling with streptavidin–horseradish peroxidase was developed with diaminobenzidine. Counterstaining was done with methyl green dye. The slides were examined and photographed using a digital camera, attached to Olympus CX51 light microscope (China) and connected to the computer. Three representative images of each slide were examined by an experienced reader. Ki-67 positive hepatocytes and apoptotic hepatocytes in liver sections were quantitated by counting the number of positive cells in 10 random microscopic fields (40×). 2.7.2. Electron microscopic study The electron microscopic study was done at King Fahd Research Center in King Abdulaziz University. Approximately 0.25 mm thick liver tissue slices were fixed in 5% glutaraldehyde at a pH of 7.25, then post-fixed by 2% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in graded ethanol and embedded in Spur epoxy resin. Thin (0.2–1.0 ␮m) and ultrathin (pale gold, 40–50 nm) sections were cut with an LKB BROMA 8800 ULTRATOM III (Sweden) ultramicrotome using EMCORP diamond knives (USA). Ultrathin sections were collected on copper grids and stained with 4% uranyl acetate in 50% ethanol followed by 0.3% lead citrate (Reynolds, 1963). The stained ultrathin sections were examined with a Philips CM 100 (Holland) transmission electron microscope at 60 kV. 2.8. Biochemical analysis 2.8.1. Determination of protein (Smith et al., 1985) The standard curve was constructed by preparing bicinchoninic acid BCA protein standard ranging from 0 to 1000 ␮g/ml by making serial dilutions (Bicinchoninic Acid Protein Assay Kit, Product Code BCA1 AND B 9643). The sample was prepared by homogenizing one volume of liver tissue (20 mg) with five volumes (100 ␮l) of the solution of (0.15 M KCl, pH 7.0) and phosphate buffer (1:9 dilution) in glass homogenizer equipped with a plastic pestle on ice (KARL Kolb B. Broun, West Germany). The samples were placed in a centrifuge at 700 × g for 10 min at −4 ◦ C (SIGMA 2-5, Germany); One part of protein sample or standard (20 ␮l) was mixed with 20 parts of the Working reagent (400 ␮l) and incubated in a water bath at 60 ◦ C. The absorbance was read at 562 nm by Kinetic microplate reader (Molecular Devices, USA). Standard curve ␮g/well vs. standard readings was plotted. 2.8.2. Determination of glycogen (Bueding and Orrell, 1964) 10 mg of the liver tissue were homogenized with 200 ␮l distilled water in a glass homogenizer equipped with a plastic pestle on ice (KARL Kolb B. Broun, West Germany). The homogenates were boiled for 5 min in a water bath (Fisher Scientific, UK) to inactivate enzymes and then placed in a centrifuge at 18,900 × g for 5 min (SIGMA 2-5, Germany) to remove insoluble material. Fifty ␮l of the Reaction Mix (46 ␮l Development Buffer, 2 ␮l Development Enzyme Mix and 2 ␮l Oxi Red Probe) were added to each well containing glycogen standard or samples. The absorbance was measured colorimetrically at 570 nm by Kinetic microplate reader

(BioTek, USA). The technique was done according to the manufacturer’s protocol (Glycogen Assay Kit. Catalog #K646-100). Calculation :

C=

Ay (␮g/ml) Sv

Ay is the amount of glycogen (␮g) in the sample from the standard curve. Sv is the sample volume (␮l) added to the sample well. 2.9. Statistical analysis Results were expressed as mean and standard deviation (SD). The statistical analysis was preformed with the Student’s t-test (in the case of normality data) and Mann–Whitney U test (in the case of non-normality) using SPSS Version 16 for Windows. P-values less than 0.05 were considered to indicate statistical significance. 3. Results 3.1. Morphometric study In the present study, the weights of the two groups was matched before the start of the experiment. Their mean weights did not record any statistically significant differences (P > 0.05). On the other hand, the mean weights of mothers fed on a low-protein diet in the last week of pregnancy showed a highly significant decrease (LP) 241.64 ± 2.29 vs. (ST) 282.67 ± 1.63 (Table 1). There was a highly significant decrease (P < 0.01) in the mean number of fetuses from mothers fed a low-protein diet (LP) 3.30 ± 4.48 vs. (ST) 11 ± 2.68. The number of corpora lutea exceeded that of the fetuses in group II (LP) (Table 1 and Fig. 1a, b). The mean value of the body weight of the fetuses from mothers of group II (LP) revealed a highly significant decrease (P < 0.01) in comparison to that of group I (ST) (Table 1). A highly significant decrease (P ≤ 0.01) in the mean weight of fetal livers from mothers fed a low-protein diet has also been observed. (LP) 0.11 ± 0.03 vs. (ST) 0.17 ± 0.14 (Table 1). The mean area (␮m2 ) of both the total hepatocytes and the cytoplasm of fetal livers from mothers fed a low-protein diet showed a highly significant increase (P < 0.01) in comparison to those fed on the standard diet. On the other hand, no statistically significant differences (P > 0.05) were recorded in the mean area (␮m2 ) of nuclei of hepatocytes in both groups (Table 1). 3.2. Histological study Light microscopic examination of sections of the fetal livers of group I (ST) using H&E stain showed indistinctly demarcated hepatic lobules, where the interlobular connective tissue was poorly developed. In the hepatic lobule, the hepatocytes were arranged as irregular, branching and interconnected cords radiating from a central vein. Blood sinusoids were seen between the hepatic cords. Hemopoietic cells were intermingled with the hepatocytes at different stages of development. The hemopoietic cells were distinguished from the hepatocytes by their rounded small spherical, darkly stained nuclei. The hepatocytes appeared rounded or polygonal in shape with ill defined cell boundaries. The cytoplasm was abundant, granular and stained acidophilic. The nucleus was euchromatic, centrally located, rounded, and contained one or more nucleoli. Some cells were binucleated (Fig. 2a). On examining sections of fetal livers from group II (LP) using H&E stain, some of the hepatocytes were stained pale as they exhibited several degrees of cytoplasmic vacuolization. Some cells showed microvesicles. Others were swollen and ballooned with larger vacuoles giving it a cobweb like appearance. The nucleus in the center of the cell was surrounded by a clear halo. Some

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Table 1 A comparison between the effects of maternal low protein diet and maternal standard diet on morphometric parameters; weight of pregnant females, number of fetuses, fetal weight, weight of fetal livers, area of hepatocytes and nuclei. A ratio of liver weight/body weight was included. N = number of female rats used. Parameter

Standard diet Mean ± SD N = 10

Body weight of females (g) at the beginning of the experiment Body weight of females in the last week of pregnancy (g) Number of fetuses per mother Body weight of fetuses (g) Weight of fetal livers (g) Liver weight/body weight Area of hepatocytes (␮m2 ) Area of nuclei (␮m2 )

209.17 282.67 11 2.0 0.17 0.17 1.20 1.00

± ± ± ± ± ± ± ±

0.75 1.63 2.68 0.42 0.14 0.14/2.0 ± 0.42 0.06 0.49

Low protein diet Mean ± SD N = 10 209.17 241.64 3.30 1.42 0.11 0.11 5.99 7.00

± ± ± ± ± ± ± ±

1.34NS 2.29** 4.48** 0.35** 0.03** 0.03/1.42 ± 0.35 0.34** 0.08NS

Values are expressed as mean ± SD. ** Highly significant (P < 0.01). *Significant (P < 0.05). NS Non significant (P > 0.05). Statistical analysis is carried out by independent student t-test.

Fig. 1. Photograph of a dissected female rat showing the fetuses in utero where (a) from group I (ST) and (b) from group II (LP). Notice the corpora lutea (black arrow), the fetus in utero (F) and the placenta (P). In (b) the uterine horns (dashed arrows) carried only one fetus.

Fig. 2. (a–c) Hematoxylin and Eosin stained sections. (a) Fetal liver of group I (ST) showing general architecture with hepatic cords radiating from the central vein (CV). Hepatocytes with abundant acidophilic cytoplasm and centrally located rounded nucleus (white arrow). Some cells were binucleated (black arrow). Notice the large number of hematopoietic cells at different stages of differentiation. (b) Fetal liver of group II (LP) showing vacuolization of the cytoplasm of the hepatocytes. Some cells showed micro vesicles (dashed arrow). Moreover the others showed either larger vacuoles (head arrow) or well defined fat droplets (black arrows). (c) Fetal liver of group II (LP) showing some hepatocytes with deeply acidophilic cytoplasm and small rounded condensed pyknotic nuclei (arrow). Blood sinusoids = (S); central vein = (CV).

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Fig. 3. (a and b) Immuno stained sections(TUNEL). (a) Fetal liver of group I (ST) with no apparent brown stained apoptotic nuclei. (b) Fetal liver of group II (LP) showing scattered apoptotic brown stained nuclei.

hepatocytes revealed well defined non-membrane bound fat droplets in the cytoplasm (Fig. 2b). Some of the hepatocytes showed signs of apoptosis in which the cells were shrunken with the cytoplasm stained bright pink and was densely eosinophilic. The pyknotic nuclei were smaller, condensed and intensely basophilic (Fig. 2c). The apoptotic cells identified in sections stained with H&E were confirmed using the TUNEL assay. There was an evident increase in the apoptotic nuclei in livers of group II (LP) in comparison to group I. (LP) 4.00 ± 1.247 vs. (ST) 1.00 ± 0.994 (Fig. 3a and b). In Oil Red O stained sections, parenchyma cells of fetal livers of group II(LP) showed increased positivity of the Oil Red O stained fat droplets compared to those in group I (ST) (Fig. 4a and b). In comparison to group I (ST), the hepatocytes in group II (LP) showed increased positivity of the PAS-positive stained glycogen granules. The glycogen infiltrated hepatocytes appeared all over the liver lobule (Fig. 5a and b). Analysis of liver cell proliferation using Ki-67 immunostaining showed that ki-67 positive hepatocytes were distributed throughout the entire lobules in both groups but decreased proliferation in fetal hepatocytes of mothers fed a low protein diet in comparison to those fed a standard diet. The mean number of Ki-67-positive hepatocytes in livers of group II (LP) was 16.66 ± 7.505 while in the case of group I (ST) it was 106.333 ± 27.353 (Fig. 6a and b). Some megakaryocytes and blood cells showed positive staining for the Ki-67 but were not counted. Using the transmission electron microscope the ultrastructure of livers was studied. Sections of fetal livers from mothers of group I (ST) showed that most of the hepatic cells had smooth cell surfaces.

Fig. 4. (a and b) Oil Red O stained sections. (a) Fetal liver of group I (ST) showing positively stained fat droplets in the cytoplasm of hepatocytes (arrow). (b) Fetal liver of group II (LP) showing increased positivity of the Oil Red O stained fat droplets in comparison to those in group I (ST).

The part of the nucleus shown had an easily identified, almost regular nuclear membrane. It contained electron-lucent euchromatin with scattered areas of heterochromatin. The cytoplasm had a granular appearance. Lysosomes and rosette shaped glycogen granules were scattered in the cytoplasm. There was a profuse amount of rough endoplasmic reticulum especially around the nuclear envelope and between the mitochondria. The cisternae of some rough endoplasmic reticulum (RER) were studded with numerous adherent ribosomes (Fig. 7a). The examined sections of fetal livers from mothers of group II (LP) revealed irregularity in the cell membranes of some hepatocytes. The nucleus had an irregular nuclear envelope. The cytoplasm contained osmiophilic fat droplets of variable sizes (Fig. 7b). In other examined hepatocytes, the nucleus appeared more regular and the cytoplasm revealed vacuolization showing an irregular cloudy appearance. Different shapes of the mitochondria were apparent. Some were rounded and others were oval or elongated in shape (Fig. 7c). Loss of mitochondrial cristae was observed in other examined grids. Occasionally, the nucleus showed compaction and margination of heterochromatin resulting in formation of sharply circumscribed masses at its periphery. The granular cytoplasm of the hepatocytes was well observed. There was also an apparent increase in the rosette shaped glycogen granules in comparison to group I (ST) (Fig. 7d). Some cells showed marked dilatation of the rough endoplasmic reticulum in addition to the swelling of some mitochondria (Fig. 7e).

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Fig. 5. (a and b) PAS stained sections. (a) Fetal liver of group I (ST) showing the positive PAS stained glycogen granules in the cytoplasm of hepatocytes. (b) Fetal liver of group II (LP) showing increased positivity of the PAS-positive stained glycogen granules (arrow) all over the hepatic lobule compared to group I (ST).

3.3. Biochemical analysis There was a highly significant decrease in the mean value of protein concentration (␮g/ml) in fetal livers from mothers of group II (P < 0.01) (LP) 4.10 ± 3.80 vs. (ST) 8.04 ± 2.41 (Table 2), whereas the mean concentration of glycogen (␮g/␮l) in fetal livers from mothers of the same group revealed a highly significant increase (P < 0.01) (LP) 0.10 ± 0.02 vs. (ST) 0.04 ± 0.01 (Table 2). 4. Discussion Previous studies indicated that offspring of rats fed a lowprotein diet during pregnancy had a higher susceptibility chronic Table 2 A comparison between the effects of maternal low protein diet and maternal standard diet on protein and glycogen concentrations in fetal livers. N = number of fetal livers examined. Parameter

Standard diet Mean ± SD N = 10

Low protein diet Mean ± SD N = 10

Protein concentration in fetal liver (␮g/ml) Glycogen concentration in fetal liver (␮g/␮l)

8.04 ± 2.41

4.10 ± 3.80**

0.04 ± 0.01

0.10 ± 0.02**

Values are expressed as mean ± SD. ** Highly significant (P < 0.01). *Significant (P < 0.05). Statistical analysis is carried out by independent student t-test.

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Fig. 6. (a and b) Immuno stained sections (Ki-67). (a) Fetal liver of group I (ST) showing proliferation of Ki-67 positively stained fetal hepatocytes. (b) Fetal liver of group II (LP) showing mild proliferation of Ki-67 positively stained fetal hepatocytes.

diseases and their mothers underwent metabolic adaptations to maintain adequate fetal development (Zambrano et al., 2005, 2006). In the present study, a significant decrease in the weight of pregnant mothers fed on a low-protein diet was noticed. Jansson et al. (2006) suggested that low maternal weight gain was due to the catabolic state and a breakdown of maternal protein and fat stores. Although the number of the corpora lutea in mothers of group II (LP) was within normal values, the number of fetuses was significantly decreased. Counting and comparing the number of corpora lutea was not included in this study yet this observation could be either due to resorption of the fetuses or failure of implantation of the blastocysts. This was attributed by Ballen et al. (2009) to the reduction in the hormones caused by protein restriction during pregnancy. The decreased levels of Luteinizing (LH) and Follicle-stimulating (FSH) hormones during 70 days in experimental groups experiencing maternal protein restriction were observed. The increase in testosterone levels at 1 year presage potential reproductive problems including changes in the ovarian cycle (Guzmán et al., 2006). Also, the weights of those fetuses revealed a significant decrease in comparison to those of group I ˜ (ST). This was in agreement with the results of Minana-Solis and Escobar (2007) who found that the body weights of pups from dams fed a low protein diet during gestation represented 55% of the body weight of pups from dams fed a standard diet during gestation. Also, Rosario et al. (2011) found that the placental leptin and insulin/IGFI, which stimulate the activity of placental amino acid transporters, were decreased in maternal protein restriction.

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Fig. 7. (a–e) Uranyl acetate and lead citrate stained ultrathin sections. (a) Fetal liver of group I (ST) showing smooth cell surface and regular well identified nuclear membrane. The cytoplasm showed a granular appearance with scattered rosette shaped glycogen granules (arrow). There were profuse amount of rough endoplasmic reticulum (RER) around the nuclear envelope and between the mitochondria (M). N = nucleus. L = lysosomes. (b) Fetal liver of group II (LP) showing lateral interdigitations of hepatic cell surfaces (arrow heads). The nucleus (N) had irregular nuclear envelope. The cytoplasm contained osmophilic fat droplets (F). (c) Fetal liver of group II (LP) showing vacuolization of the cytoplasm (black arrow). Different shapes of mitochondria were apparent (white arrow). (d) Fetal liver of group II (LP) showing loss of crestea in some mitochondria (arrow head). The nucleus showed compaction and margination of heterochromatin (white arrow). Notice the apparent increase in rosette shaped glycogen granules in the cytoplasm (black arrow). (e) Fetal liver of group II (LP) showing dilated rough endoplasmic reticulum (arrow head). Some mitochondria were swollen (M). N = nucleus.

In agreement with the present results, previous studies (ElKhattabi et al., 2003; Zhang and Byrne, 2000) have shown that the liver weights in fetuses of rat dams fed a low protein diet containing 8% protein was significantly reduced compared to those fed on a diet containing 20% protein. In this study, the mean area of hepatocytes and their cytoplasm in fetal livers from mothers of group II revealed a highly significant increase while that of the nuclei did not record any change. This could be attributed to signs of cell injury, which, in this case, would be hydropic degeneration. Histological examination of sections of fetal livers of group II (LP) stained with H&E exhibited several degrees of cytoplasmic vacuolization, ranging from micro vesicles to large size vacuoles resulting in ballooning of the hepatocytes. This was also evident at the ultrastructural level where irregular cloudy vacuolization appeared in the cytoplasm of some cells. Filho (2007) explained that the cytoplasmic vacuolization implies increased permeability of cell membranes which led to an increase in intracellular water. However, Kumar et al. (2007) stated that the cytoplasmic vacuolization represents

distended and pinched-off segments of the endoplasmic reticulum. The observed apoptotic hepatocytes in fetal livers of group II (LP) suggests that maternal low protein diet during pregnancy plays a crucial role in promoting programed cell death in fetal liver. This was emphasized by DNA fragmentation occurring as one of the final stages of cell death, and has long been considered a hallmark of apoptosis and one of the defining biochemical events of the pathway. In the present study, apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The number of apoptotic nuclei increased in fetal livers of group II (LP) in comparison to sections of group I(ST). (LP) 4.00 ± 1.247 vs. (ST) 1.00 ± 0.994. Similarly Kidney TUNEL performed by Tafti et al. (2011) showed apoptotic nuclei significantly increased in kidneys of newborn of undernourished mothers. Welham et al. (2002) observed in their study that protein restriction in pregnancy was associated with increased apoptosis. In addition, Kumar et al. (2007) found that nutritional deficiencies remain a major cause of cell injury. They described the injured cell to have increased eosinophilia, nuclear shrinkage, fragmentation

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with small masses of the condensed heterochromatin distributed throughout the nucleus. This was in agreement with the present ultra structural results where some nuclei of fetal hepatocytes in group II (LP) showed compaction and margination of heterochromatin resulting in formation of sharply circumscribed masses at its periphery. Pedrycz et al. (2005) described two main ways of apoptotic signal transmission in mammal cells: external and internal (mitochondrial) way. The internal path is a response to factors causing DNA damage, and the process takes place mostly in mitochondria, most often in connection with proapoptotic proteins from BCL-2 group, including BAX protein. The increased Oil Red O stained fat droplets in the liver parenchyma in of fetuses of group II (LP) was confirmed at the ultrastructural level where osmiophilic fat droplets of variable sizes appeared in the cytoplasm. Also Cheng et al. (2009) showed that fatty acid reacts with osmium tetroxide, imparts lipid droplets an electron density that reflects fatty acid. In agreement with this finding Erhuma et al. (2006) noticed that, maternal low protein diet causes macrovesicular steatosis in livers by appearance of fat droplets. Also Souza-Mello et al. (2007) suggested that, in maternal protein restriction, hepatic steatosis can be induced in rat fetuses. Park et al. (2003) suggested that as lipid homeostasis is mainly dependent on the liver, the underlying lipid deregulation in nutritionally restricted rats would be mediated partly through alterations of liver structure. Wei et al. (2006) tried to explain the mechanisms linking lipid accumulation to cell death. They found that an accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. They also demonstrated that saturated fatty acids disrupt endoplasmic reticulum homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation. In the current study, the hepatocytes of fetal liver of group II (LP) showed increased positivity of the PAS-positive stained glycogen granules appearing all over the liver lobule. This was revealed as an apparent increase in the rosette shaped glycogen granules occupying the cytoplasm of hepatocytes by the electron microscope. The biochemical results of this study also revealed an increase in the glycogen concentration in the liver of fetuses of group II (LP). In accordance with the above results, Gosby et al. (2003) found that offspring of rats fed on a low protein diet had increased hepatic glycogen. They explained that the increase in glycogen storage was not because of an increase in glycogen synthesis but due to increased sensitivity to insulin despite reduction in its secretion. While Li et al. (2007) hypothesized that increased liver glycogen in fetuses of the nutrient restricted mothers suggests energy conservation by reduction of glycolysis or increased gluconeogenesis in response to reduced fetal nutrients. But, the maternal plasma glucose was maintained and even showed a tendency to rise which would help to maintain fetal glucose. According to Hilakivi-Clarke et al. (1999) the increase in glycogen content is attributed to an increase in insulin receptors in the liver. Ki-67 labeling has gradually supplanted other techniques previously used to evaluate proliferation rates (Klein et al., 2002). Our results are consistent with El-Khattabi et al. (2003) who studied the effect of maternal LP diet on fetal hepatocyte proliferation. They found that the DNA content, as measured by the DAPI method, was significantly reduced by 30% in the LP group compared with control values. Li et al. (2009) found that the mitotic rate in hepatic cells indicated by Ki67 in nutrient restricted baboons has also been decreased. Higami et al. (2000) suggested that the suppression of the proliferation of hepatocytes under dietary restriction, resulting in their reduced number during development may be mediated by overexpression of the p53 suppressor gene. The hypothesis presented by Gruppuso et al. (2005) that restricted nutrient availability sends signals toward the hepatocyte cell cycle in fetuses of fasted mothers. It accounts for decreased hepatocyte proliferation and

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liver mass. This was based on the association of the reduced arginine in fetal hepatocyte (19th day) with decreased DNA synthesis. They found that low arginine induced changes in the translational machinery. That was an indication of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin. In the present work the ultra structural study of fetal livers from mothers of group II (LP) revealed irregularity in the cell membrane and nuclear envelope. The irregularity of both cell and nuclear membranes were signs of necrosis according to Kumar et al. (2007). However, no apparent disruption of the membranes could be revealed in the present study indicating that the injury to the cells did not lead to their necrosis. Galluzzi et al. (2007) stated that necrotic cells are characterized by discontinuities in plasma and organelle membranes and profound nuclear changes culminating in nuclear dissolution. In the present work, nuclei of some hepatocytes showed compaction and margination of heterochromatin resulting in the formation of sharply circumscribed masses at its periphery without profound nuclear changes culminating in nuclear dissolution. Different shapes, swelling and loss of cristae of the mitochondria in addition to the dilatation of the rough endoplasmic reticulum cisternae were apparent on examination of fetal livers from mothers of group II (LP). Magwere et al. (2006) noticed that dietary restriction only alters mitochondrial morphology (size, shape, and number). But, Morin et al. (2001) explained that mitochondrial swelling may be due to lipid peroxidation that promotes the permeability transition causing mitochondrial swelling and calcium release. Furthermore, Kumar et al. (2007) suggested that increased permeability of the mitochondria may result in leakage proteins by apoptosis. Dirlik et al. (2009) found that apoptotic changes in hepatocytes are accompanied with dilatation of the rough endoplasmic reticulum which could explain the similar results observed in this study. Wei et al. (2006) also indicated that protein restriction in early life causes long-lasting changes in mitochondria, and this change is more evident in the liver and skeletal muscle that may contribute to the development of insulin resistance in later life. In the present study, the biochemical analysis revealed a significant decrease in the protein concentration in fetal livers from mothers of group II. On the other hand there was an increase in the concentration of glycogen. This biochemical analysis was compatible with the histological results.

5. Conclusions A diet containing 5% protein is still able to support pregnancy, but produces an unusual pattern of fetal growth compared with a control diet, containing the optimum of 20% protein. Overall, this morphometric and histological study showed that the hepatocytes of rat fetuses from mothers fed a low protein diet during pregnancy exhibited apoptosis, steatosis and decreased proliferation. The biochemical study also confirmed the above results where the hepatic concentration of glycogen was increased and that of the proteins was decreased. It thus appears that maternal metabolic condition not only reduces fetal growth in response to protein restriction, but also altered the structure of the liver.

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