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Keywords:

  • malnutrition;
  • lactation;
  • endometrium;
  • collagen;
  • angiogenesis;
  • estradiol

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The aim of this manuscript was to evaluate the effects of maternal protein-energy-restriction and energy restriction during lactation on endometrial collagen and blood vessels, uterus Erα expression, and estradiol serum levels in the rats offspring at puberty. At parturition, dams were grouped as: control group (C), with free access to standard rat chow containing 23% protein and 17,038.7 KJ/Kg; protein-energy restricted group (PER), with free access to formulated chow containing 8% protein but made isoenergetic to the C diet (17,038.7 KJ/Kg); and energy-restricted group (ER), which received standard rat chow containing 23% protein based on the mean ingestion of the PER group corresponding to 60% of that consumed by the control group. After weaning, all female pups had free access to standard laboratory chow until puberty, when they were killed at the diestrum stage. The uterine ERα expression was determined by Western-Blot and estradiol serum levels by radioimmunoassay. Endometrial collagen and blood vessels were quantified by stereology. The volumetric density of blood vessels (C = 70.7 ± 2.2; PER = 29.2 ± 2.4; ER = 32.3 ± 3.6; P < 0.001) and endometrial collagen (C = 31.1 ± 1; PER = 26.9 ± 1.0; ER = 26.5 ± 0.7; P < 0.05) were significantly reduced in both malnourished groups. The ER group presented higher estradiol serum levels (C = 69.2 ± 6.4; PER = 73.4 ± 5.5; ER = 101.0 ± 5.4; P < 0.01) in relation to C and PER groups. ERα expression was greater in both malnourished groups (C = 0.11 ± 0.02; PER = 0.41 ± 0.12; ER = 0.35 ± 0.03; P < 0.05). In conclusion, maternal malnutrition during lactation caused changes in endometrial angiogenesis, collagen deposition, and Erα expression in female offspring that will appear in puberty and could affect the reproductive biology of the female offspring. Anat Rec, 2010. © 2009 Wiley-Liss, Inc.

The extracellular matrix (ECM) is considered to play an important role in the stability of tissues and in the regulation of growth and differentiation of cells (Iwahashi et al.,1997). Collagen, a component of the extracellular matrix, plays a role in several uterine processes, such as pregnancy, trophoblast invasion, and postpartum involution (Manase et al.,2006).

The rat model has been used to study the activities of the uterus, such as involution, menstruation, pregnancy, cervical dilation, and implantation (Helvering et al.,2005). In these processes, the extracellular matrix components remodeling is essential and a family of enzymes, the matrix metalloproteinases (MMPs), promotes degradation of extracellular matrix, particularly collagen, and allow tissue reorganization (Kelly et al.,2003; Manase et al.,2006).

Some papers have investigated the relationship between extracellular matrix turnover and estradiol in humans and animals models, showing that estradiol induces the expression of MMP-7 and MMP-14 and reduces the expression of metalloproteinases inhibitor genes (TIMP-3) in rat uterus (Helvering et al.,2005). In uterus gene array, the expression of mRNA for MMP-2 was up-regulated slightly by estradiol and 5-week treatment of ovariectomized rats with estradiol, which significantly increased MMP-2 activity in uterine explants (Cox and Helvering,2006).

Moreover, several authors have shown that a correlation exists between ERα expression and collagen concentration. According to Lydrup and Ferno (2003), this regulation seems negative, because in their study collagen concentration was lower in human uterine arteries with higher ERα expression. Rodriguez et al. (2003) also showed a negative correlation, evaluated by collagen birrefringency intensity and subepithelial ERα expression on pregnant guinea-pig cervix. Therefore, it appears that both estradiol serum levels and ERα can regulate tissue collagen concentration, although in a different way.

Blood vessels are also considered to play an important role in several uterine functions. Successful embryo development requires an extensive endometrial angiogenesis in proximity of the implantation site (Berndt et al.,2006). Angiogenesis is a fundamental process by which new blood vessels are formed and it is essential in reproduction, development, and wound repair. Angiogenesis, involving proliferation, maturation, and migration of endothelial cells is not only regulated by ovarian steroids but also by angiogenic growth factors (Srivastava et al.,1998), that is, basic fibroblastic and vascular endothelial growth factors (Pavelock et al.,2001).

Extensive epidemiological and laboratory evidence indicates that a suboptimal environment during fetal and neonatal development in both humans and experimental animals impacts on offspring susceptibility to develop several disturbances in the adulthood, such as obesity, vascular disease, diabetes mellitus type II, and thyroid function (Barker and Osmond,1986; Ramos et al.,1997; Barker,1998; Poston,2007).

Recently, we have shown that female offspring whose mothers were submitted to protein and energy malnutrition only during lactation, the onset of puberty was delayed, in spite of a normal estrous cycle and the atrophied uterine endometrial glands (da Silva Faria et al.,2004; Brasil et al.,2005).

The aim of this study was to evaluate if maternal malnutrition during lactation can affect the endometrial collagen, blood vessels, Erα expression, and estradiol serum levels.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Protocol

Wistar rats were kept in a room with controlled temperature (25 ± 1°C) and with artificial dark–light cycle (lights on from 7:00 AM to 7:00 PM). Three-month old, virgin female rats were caged with one male rat at a proportion of 2:1. After mating, each female was placed in an individual cage with free access to water and food until delivery. The use and handling of experimental animals followed the principles described in the Guide for the Care and Use of Laboratory Animals (Bayne,1996) and the project was approved by the local Ethical Committee for care and use of laboratory animals.

Nine dams were randomly assigned to one of the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein; protein-energy restricted group (PER), with free access to an isoenergy and protein-restricted diet containing 8% protein and the energy-restricted group (ER), receiving standard laboratory diet in restricted quantities, which were calculated, each day, according to the mean ingestion of the PER group and corresponded to 60% of that consumed by the control group. The PER group, in spite of having free access to diet, consumed about 60% of that consumed by the control group (Passos et al.,2000). In this way, the amount of food consumed in both ER and PER groups was almost the same and measured each day.

The low-protein diet was prepared in our laboratory and its composition is shown in Table 1. Vitamins and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets (Reeves et al.,1993). Within 24 hr of birth, excess pups were randomly removed so that only 6 pups were kept per dam, as it has been shown that this procedure maximizes lactation performance (Fischbeck and Rasmussen,1987). Malnutrition of the studied rats was started at birth, which was defined as day 0 of lactation (day 0), and was ended at weaning (day 21). After weaning, female pups of the same treatment group were housed in groups of three animals per cage, and given unlimited access to food and water until puberty (day 40). Then, only the animals at the diestrum stage were sacrificed with a lethal dose of pentobarbital.

Table 1. Composition of control and protein-restricted diets
 ControlaProtein- Restrictedb
  • a

    Standard diet for rats (Nuvilab-Nuvital ltd., Paraná, Brazil).

  • b

    The protein-restricted diet was prepared in our laboratory by using the control diet, with replacement of part of its protein content with cornstarch. The amount of the latter was calculated to replace the same energy content of the control diet.

  • c

    The principal protein resources are soybean wheat, steak, fish, and amino acids.

  • d

    Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets (Reeves et al.,1993).

Ingredients (g/Kg)  
 Total Proteinc230.080.0
 Corn starch676.0826.0
 Soybean oil50.050.0
 Vitamin mixd4.04.0
 Mineral mixd40.040.0
Macronutrient composition (%)  
 Protein23.08.0
 Carbohydrate66.081.0
 Fat11.011.0
Total energy (KJ/Kg)17,038.717,038.7

To evaluate the nutritional state, food consumption of the offspring was monitored each day from weaning onwards, whereas body weight and linear growth (nose-tail) were monitored every 5 days from birth until the end of experiment. The blood was collected by cardiac puncture and the serum kept at −20°C for subsequent determination of hormonal parameters. Uterus was excised, dissected, weighed, and then separated into two horns. To avoid contamination with ovary or vaginal tissue, only the medium section of each horn was used. The right horn was kept at −80°C for subsequent measurements of estrogen receptors α by Western blot. The left horn was processed by routine methods and embedded in paraffin. Uterine sections were systematically random sampled, 5 μm in thickness, and stained with Gomori's trichrome (Bradbury and Rae,1996) and Picrosirius Red in polarizations microscope to show the different possible types of collagen in the samples. The analyzed fields were digitized with 100× final magnification using a video camera coupled to a light microscope.

Stereological parameters.

Some samples were stained with hematoxylin and eosin to check the integrity of the tissue. Line and point probes are obtained in a field by superimposing a grid consisting of an arrangement of lines and points (Cruz-Orive and Weibel,1990; Mandarim-de-Lacerda,2003). The M42 multipurpose test system was used where only the structures not crossing the test system forbidden line were considered. The short-line length (d) was used to calibrate the test system, the line length Lt is 21d, the test area AT is 36.36d2, and it has 42 test points (Pp). Only the endometrial compartment of the uterus was quantified in this study (Brasil et al.,2005). From each uterus, five different sections were selected from five fragments. Then, five random fields were evaluated from each section. Therefore, there were 25 test areas from each uterus. The stereological parameters analyzed were: volumetric density (Vv) of the endometrial collagen and volumetric density (Vv) of the endometrial blood vessels (Vv = Pp/Pt (%), where Pp is the number of test points in the structure and Pt is the number of total test points). All blood vessels (independent of the type) that were seen in the 100× magnification were counted in this study.

Western blot.

Uterine horn were homogenized in 500 μL TEG buffer (50 mM TRIS pH 7.4, 1.5 mM EDTA, 50 mM NaCl, Glycerol 10%, 5mM DTT, 10μg/mL leupeptin). The homogenate was centrifuged at 100,000g for 120 min at 4°C. The proteins extracted form the supernatants were measure using Bradford's method (Bradford,1976) and analyzed by Western blot technique. 80 μg of total protein was loaded in each lane of 8% polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membrane, and the detection of specific proteins was performed using ERα-specific antibody (Mouse monoclonal 200 μg/mL Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase IgG was used as a secondary antibody, followed by autoradiography using ECL detection reagents supplied by Amersham (Braunschweig Germany). Bands were computer-scanned and their relative intensities determined by densitometry using Scion Image Software.

Steroid determination.

The estradiol and testosterone serum concentrations were determined using a specific radioimmunoassay for each hormone (ICN Pharmaceuticals, CA). The intra- and inter-assay variation coefficients were 4.6 and 7.5% for testosterone and 6.4 and 5.9% for estradiol. Sensitivity of the radioimmunoassay was 0.03 ng/mL for testosterone and 7.4 pg/mL for estradiol (Teixeira et al.,2007).

Statistical Analysis

The data were reported as mean ± standard deviation of five animals. Statistical significance of experimental observations was determined by the ANOVA, followed by Newman Keuls pos test (Sokal and Rohlf,1995). Stereological data were analyzed by the Kruskal-Wallis test, followed by Dunn's post test. The level of significance was set at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Figure 1 shows food consumption (A), body weight (B), and linear growth (C) of the groups throughout the experiment. Food consumption of the offspring was evaluated from weaning until sacrifice. Both PER and ER groups had lower food intake than the C group (P < 0.001) from weaning until the end of the experiment (Fig. 1A). Overall, at each time point of measurement from day 4 after birth until day 40, the body weight of both PER and ER groups was significantly lower when compared with controls (P < 0.001) (Fig. 1B). Similarly, linear growth showed the same pattern, that is, PER and ER groups had significantly lower linear growth (P < 0.001) when compared with controls from day 6 after birth until day 40 (Fig. 1C).

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Figure 1. Food consumption (A), body weight (B) and linear growth (C) of control group (C), protein-restricted group (PER), and energy restricted group (ER) from birth to 40 days of age. Values are given as mean ± standard deviation of five animals.

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Figures 2 and 3 show that both PER and ER groups presented lower absolute and relative uterine weight when compared with C group (C = 0.27 ± 0.02; 2.01 ± 0.21 PER = 0.15 ± 0.006; 1.46 ± 0.07 ER = 0.16 ± 0.004; 1.56 ± 0.06; P < 0.001 and P < 0.05, respectively).

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Figure 2. Absolute uterine weight in control group (C), protein-restricted group (PER), and energy restricted group (ER). Values are given as mean ± standard deviation of five animals.*P < 0.001 vs. C.

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Figure 3. Relative uterine weight in control group (C), protein-restricted group (PER), and energy restricted group (ER). Values are given as mean ± standard deviation of 5 animals.*P < 0.05 vs. C.

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Stereological quantification revealed that there was a significant reduction in the volumetric density of blood vessels in both PER and ER groups, when compared with C group (C = 70.7 ± 2.2; PER = 29.2 ± 2.4; ER = 32.3 ± 3.6; P < 0.001) (Fig. 4A). The volumetric density of endometrial collagen was also lower in both PER and ER groups, when compared with C group (C = 31.1 ± 1.0; PER = 26.9 ± 1.0; P < 0.05; ER = 26.5 ± 0.7; P < 0.01) (Fig. 4B).

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Figure 4. Volumetric density of endometrial blood vessels (A) and endometrial collagen (B) in control group (C), protein-restricted group (PER), and energy restricted group (ER). Values are given as mean ± standard deviation of 5 animals.*P < 0.001 vs. C; #P < 0.05 vs. C; **P < 0.01 vs. C.

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As regards the hormonal serum levels, only the ER group showed a significant alteration. The estradiol serum level was higher in the ER group when compared with both PER and C groups (C = 69.2 ± 6.4; PER = 73.4 ± 5.5; ER = 101.0 ± 5.4, P < 0.01) (Fig. 5A). ER group presented lower testosterone serum levels when compared with both PER and C groups (C = 0.09 ± 0.02; PER = 0.10 ± 0.02; ER = 0.03 ± 0.008, P < 0.05) (Fig. 5B).

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Figure 5. Estradiol (A) and testosterone (B) serum levels in control group (C), protein-restricted group (PER), and energy restricted group (ER). Values are given as mean ± standard deviation of five animals. *P < 0.01 vs. C; #P < 0.01 vs. PER; **P < 0.05 vs. PER and C.

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Figure 6 shows that ERα expression was significantly higher in both PER and ER groups when compared with C group (C = 0.11 ± 0.02; PER = 0.41 ± 0.12; ER = 0.35 ± 0.03, P < 0.05).

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Figure 6. Uterine ERs expression in control group (C), protein-restricted group (PER), and energy restricted group (ER). Values are given as mean ± standard deviation of five animals. *P < 0.05 vs. C.

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The analysis of histological sections by polarization microscopy revealed that there was a difference in the type of collagen in the endometrium of the three groups. The C group presents a predominance of red color, while both PER and ER groups present a predominance of green color (Fig. 7).

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Figure 7. (A) Histological section of uterus by polarization microscopy in control group (C). Final magnification ×100. (B) Histological section of uterus by polarization microscopy in protein-restricted group (PER). Final magnification ×100. (C) Histological section of uterus by polarization microscopy in energy restricted group (ER). Final magnification ×100.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Some authors have shown that lactation could be a critical period in determining the future endocrine status of the progeny (Moura et al.,1997; Ramos et al.,1997). The maternal nutritional state during lactation is equivalent and possibly even more important than that during gestation, as evidenced in a study by Leonhardt et al. (2003), which showed that the offspring whose dams were malnourished during lactation had more drastic consequences on gonadal development when compared with the offspring whose dams were malnourished only during pregnancy or during pregnancy and lactation. Guzman et al. (2006) showed similar results. Based on these papers, we decided to assess the effects of malnutrition only during the lactation period.

Recently, we showed that maternal protein and energy malnutrition during lactation leads to growth retardation and a delay in the onset of puberty in female pups (da Silva Faria et al.,2004). Also, protein and energy restriction during lactation leads to an atrophy of the uterine endometrial glands of the offspring at puberty (Brasil et al.,2005).

All alterations described in this article regarding the effects of maternal malnutrition during lactation on the uterus function of female offspring at puberty could possibly be related to changes in food intake, body weight, or linear growth. The food intake of both PER and ER groups was lower throughout the experiment; however, it did not reach the control group values. Both restricted groups presented low body weight and linear growth from the first few days of life until the end of the experiment, even after having free access to normal diet containing 23% protein at weaning. Therefore, it seems that maternal malnutrition programs and the food intake of the offspring being responsible for the reduction observed in the weight gain and linear growth.

In this article, we show that only the offspring whose mothers were submitted to energy malnutrition during lactation presented an increase in the estradiol serum levels. Previous results from our group (Passos et al.,2000) showed that the milk of mothers submitted to energy malnutrition during lactation presented high lipid levels, while protein-energy malnutrition mother's milk had no alteration. We can assume that the high lipid concentration in the milk of ER group could result in a stimulus to estradiol synthesis that continued higher even after the normalization of food intake after weaning, characterizing a metabolic programming of the ovary.

The rat uterus, a major target tissue for ovarian steroids, has served as an excellent model for studying hormonal regulation of ERα expression. Both, up-regulation and down-regulation of ERα by estradiol has been reported in the rat uterus, depending upon the physiological state of the animal and/or the experimental system used (Manni et al.,1981; Rosser et al.,1993; Zhou et al.,1993; Zhou et al.,1995; Wang et al.,1999; Nephew et al.,2000). Our results show that both treated groups presented an increase in ERα expression, despite the differences in the estradiol serum levels. We believe that differences in the physiological state of the animals as a result of the protein and energy dietary treatments could have contributed to the loss of ERα autoregulation by estradiol in those animals.

No relationship between estradiol levels and blood vessels was observed in the offspring of mothers submitted to maternal malnutrition during lactation. Both dietary treated groups presented a reduction in the volumetric density of blood vessels despite the differences in the estradiol serum levels. Although several papers have demonstrated that angiogenesis is influenced by estradiol, it is known that angiogenic growth factors such as basic fibroblastic growth factors (Srivastava et al.,1998) and vascular endothelial growth factors (Pavelock et al.,2001) can also regulate angiogenesis.

Khorram et al. (2007) have demonstrated that 50% maternal food restriction from day 10 of gestation to term, led to reduced number of mesenteric branching and renal medullary microvessels in the 1-day-old undernourished newborns. Endothelial cells from maternal restriction offspring generated shorter neovessels in culture compared with controls. In addition, the authors found a significant decrease in VEGF protein expression in mesenteric microvessels and aortas in 1-day-old offspring. These results suggest that maternal food restriction results in inhibition of VEGF expression in microvascular and aortic endothelial cells, resulting in decreased angiogenesis.

Maternal malnutrition caused an increase in uterine ERα expression and lower volumetric density of endometrial collagen in both dietary treated groups. In the uterus, ER α is the ER predominant isoform (Wang et al.,1999) and some authors have demonstrated a negative correlation between ERα expression and collagen (Lydrup and Ferno,2003; Rodriguez et al.,2003) which is in agreement with our results.

As showed by Junqueira et al (1979), one outcome of the Sirius Red-polarization method is the observation that sites that contain collagen type I, when studied with polarization microscopy, show a different color when compared with regions that present collagen type III. Although this stain may not be reliable to determine all the different types of collagen, it is possible to suppose that the difference in the color among the groups is indicative of the predominance of a specific type of collagen over the other.

Teodoro et al. (2003) demonstrated that collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant rats. Collagen III is a fibrillar collagen with known roles in differentiation and migration (Olsen and Ninomiya,1999) and, together with collagen I, form the structural support for the endometrium during the establishment of pregnancy (Teodoro et al.,2003). Pioneer studies (Birk et al.,1988); (Fleischmajer et al.,1990a,b) have suggested that collagen type I is the main structural component of fibrils, while other types of collagens (III or V) regulate fibril assembly and thickening.

During embryo implantation in the mouse, an unusually rapid collagen fibril thickening occurs in the decidua whereas in the nondecidualized area, close to the myometrium, the fibrils remain thin throughout pregnancy (Zorn et al.,1986; Alberto-Rincon et al.,1989). Based on these results, we can suggest that changes in the uterus collagen types of females whose mothers were subjected to malnutrition during lactation could alter the decidualization and embryo implantation. Also, these alterations could be related to the reduction in the fertility rate showed in the one-year-old rats whose mothers were malnourished during pregnancy and/or lactation periods (Guzman et al.,2006). In agreement with this hypothesis, we recently showed that the animals whose mothers were submitted to protein-energy restriction present a reduction in a Graaf follicle number related to a lower expression of androgen and estrogen receptor genes (da Silva Faria et al.,2008).

Data from epidemiological as well as in vivo animal studies have given rise to the concept of developmental programming whereby the quantity or quality of nutrition at the perinatal periods has permanent consequences for later life (Passos et al.,2000; Teixeira et al.,2002; Heywood et al.,2004; Guzman et al.,2006). In conclusion, maternal malnutrition during lactation programmed the food intake, body weight, and uterus function leading to changes in the endometrial ERα expression, angiogenesis, and collagen types. These alterations include uterine functions and several processes needed for embryo development, as implantation, nutrition, and maintenance of pregnancy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors are grateful to Richard Medeiros, Rouen University Hospital Medical Editor, Rouen, France for his valuable help in editing the manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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