Introduction

Zinc (Zn) is a constituent of various enzymes and proteins as well as an important regulator of biological functions. Zn deficiency can lead to dermatitis, alopecia, decreased growth, and impaired immune function [1]. Zn also plays important roles in bone formation activity. Animal studies have shown that Zn deficiency results in low bone mass [2, 3], and inadequate intake of Zn via diet results in a decrease in the number of osteoblasts [4] and chondrocytes [5] in animal bone. In humans, low serum Zn concentration and decreased bone turnover have been reported in small-for-gestational age preterm infants [6]. In addition, low Zn intake has been reported to be associated with low bone mass in adult women [7]. Studies in osteoblastic cell cultures have shown that Zn stimulates DNA synthesis in osteoblast-like cells [8] and has an inhibitory effect on bone resorption [9].

Although consequences of severe Zn deficiency during pregnancy have been examined in a number of animal models, such nutritional status is uncommon in human populations. However, marginal Zn deficiency is now recognized as prevalent in both industrialized and developing countries [10]. Despite the likely high prevalence of maternal Zn deficiency, data supporting maternal Zn deficiency as a cause of poor fetal growth are far from conclusive. One human study has been published suggesting that maternal Zn deficiency lowers fetal bone growth [11]. In the rhesus monkey, the Zn status of bone in newborns from dams fed marginally Zn-deficient diets during pregnancy was reported a quarter century ago [12]. Nevertheless, the involvement of Zn status during pregnancy on neonatal bone metabolism has remained unclear until now. The aim of the present study was to investigate the influence of marginal Zn deficiency during pregnancy on bone matrix formation of newborn rats using specific parameters of Zn status and bone metabolism in neonates and dams.

Materials and Methods

Diets

Rats were fed an egg-white-based experimental diet that included other compositions (Table 1) based on the AIN-93G formulation [13]. The diets contained 35 mg Zn/kg (normal control, N) and 7 mg Zn/kg (marginally Zn deficient, L), as Zn carbonate. The Zn content of both diets was confirmed after wet ashing by inductively coupled plasma atomic emission spectroscopy (ICP-AES; ICPS-7510, Shimadzu, Tokyo, Japan).

Table 1 Composition of Experimental Diets (g/kg diet)
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Experimental Design

The experiments were performed in accordance with the guidelines of the ethics committee on animal use of Meiji Dairies Corporation and Law No. 105 and Notification No. 6 of the Government of Japan. Virgin Sprague-Dawley female rats (purchased from Japan SLC, Hamamatsu, Japan) at 9 weeks of age were housed in a temperature-controlled room with a 12:12-h light–dark cycle and free access to water and a commercial diet for a 7-day acclimation period. The rats (n = 14/diet group) were randomly assigned to N-diet or L-diet groups. After 14 days with the experimental diets, individual female rats were housed together with a single male rat for 1 day to mate; both rats were fed the same diet. After mating, the female rats were maintained with the same experimental diet. Throughout the experiment, food intake and animal weight were recorded every 3 days except for on the day of mating. On 20 days postconception, the pregnant rats were anesthetized with ethyl ether and sacrificed with aortic puncture. Pups (neonates) were aseptically delivered by cesarean section. The litter size, neonate weight, and placenta weight were recorded. The time point of cesarean section was randomized between groups. Neonates were sacrificed by decapitation. All the littermate serum samples from each dam were pooled to one serum sample. Serum samples were maintained at −80°C until analysis of various parameters.

Tissue and Serum Mineral Analyses

A carcass of randomly chosen littermate from each dam was homogenized, then wet-ashed with ultrapure nitric acid. A right femur and whole calvaria of another randomly selected littermate from each dam was wet-ashed with ultrapure nitric acid. Wet-ashed samples were measured for Zn, phosphorus, and calcium concentrations with ICP-AES. Serum mineral concentrations were measured using commercial kits (Zn: Zn Test Wako and calcium: Ca E-Test Wako, Wako Pure Chemical Industries, Osaka, Japan; inorganic phosphorus with Fuji Dry Chem Slide IP-P, Fuji Film Industries, Tokyo, Japan).

Assessment of Bone Matrix Formation Using Calvarial Organ Culture

Calvarial organ culture [14] was performed to evaluate bone matrix formation activity in one neonate randomly selected from randomized 12 dams in each diet group. Briefly, aseptically dissected and cleaned neonatal half calvariae were placed concave side down on the sterile plastic grid in 12-well tissue culture plates and cultured for 7 days with BGJb medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1 mg/mL of bovine serum albumin, 100 U/mL each of penicillin/streptomycin, 0.092 mg/mL of glutamine, and 50 μg/mL of ascorbic acid (basal medium). The other set of calvaria halves was divided into two groups and cultured in medium with added basal medium and 20 μmol/L Zn (as Zn sulfate) to investigate the direct and compensatory effect of Zn on bone matrix formation (n = 6/group) or with 100 nmol/L of corticosterone to confirm integrity of the assay system, presuming that negative bone matrix formation would be observed (n = 6/group). The cultured calvariae were fixed in 10% formalin-containing phosphate-buffered saline and decalcified overnight in 14% EDTA containing 10% buffered formalin. The next day, the demineralized calvariae were processed overnight using the following protocol: 70% ethanol for 45 min, twice with 95% ethanol for 45 min, four times with 100% ethanol, twice with xylene, and twice with paraffin for 45 min. The processed calvariae were embedded in paraffin blocks. Three different 5-μm-thick sections, which included the coronal suture area at 400-μm, 800-μm, and 1,200-μm depths, were cut parallel to the midline suture. The sections were mounted onto silane-treated glass microscope slides, deparaffinized, and stained for collagen fiber with Masson’s trichrome staining. With this staining method, the original bone area was stained darker, whereas new bone appeared as a lighter color. With a charge-coupled device camera under microscopy, three images per section were captured, followed by measurement of new bone area with the software program ImageJ (NIH, Bethesda, MD, USA). New bone area was calculated as percentage of total bone area.

Biochemical Parameter Analyses

Serum osteocalcin concentration was analyzed with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Rat-MID Osteocalcin ELISA; Nordic Bioscience Diagnostics a/s, Herlev, Denmark). Serum corticosterone level was measured using an RIA kit (COAT-A-COUNT Rat Corticosterone; Diagnostic Products Corporation, Los Angeles, CA, USA). Serum insulin-like growth factor (IGF)-I level was measured using an ELISA kit (IGF-I HS ELISA; Immunodiagnostic Systems Ltd., Boldon).

Statistics

Data are presented as means ± SE. Statistical analysis was performed with Student’s t test using the software program StatView (SAS Institute Inc., Cary, NC, USA), except for the in vitro bone matrix formation assay. Two-way analysis of variance (ANOVA) was performed for the in vitro bone matrix formation assay using the effects of dam diet and Zn addition; these effects were applied to all groups, except the groups receiving corticosterone addition. In every statistical analysis, a P value of <0.05 was regarded as significantly different.

Results

Growth Parameters of Dams and Neonates

Daily food intake and final body weight of dams were not significantly different between the two dietary groups. Litter size and body weight for the neonates were comparable in both groups. Relative placental weight for neonates was not different between the two groups (Table 2).

Table 2 The Effect of Maternal Dietary Zinc Level on Growth Parameters for Dams and Neonates
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Mineral Status of Neonates

Zn content was about 20% lower in carcasses and about 30% lower in the femurs and calvariae of neonates from L-diet-fed dams than those from N-diet-fed dams. Phosphorus content was significantly lower only in femurs of neonates from L-diet-fed dams compared with those from N-diet-fed dams; however, the content was comparable in the carcasses and calvariae between the two diet groups. The calcium content of neonatal carcasses, femurs, and calvariae was not different between the dam groups (Table 3). Serum Zn concentration was lower in neonates from L-diet-fed dams than that from dams fed on N diet (P < 0.0001). However, phosphorus and calcium concentrations for neonatal serum were not significantly different between the two groups (data not shown).

Table 3 The Effect of Maternal Dietary Zinc Levels on Mineral Content of Pups
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Bone Matrix Formation in Cultured Neonatal Calvaria

Two-way ANOVA for four groups of culture medium alone and culture medium + Zn demonstrated clear impairment of bone matrix formation in the cultures from the L-diet-fed dam group (P = 0.002). The addition of 20 μmol/L Zn to the culture medium, which is a normal serum concentration for adult rats, had no effect on bone matrix formation for the calvariae of neonates from N-diet dams. Addition of 100 nmol/L corticosterone to culture media, the normal serum concentration in pregnant rats [15], resulted in a clear reduction in bone matrix formation in both groups (Fig. 1).

Fig. 1
figure 1

The effects of maternal dietary zinc (Zn) levels and acute Zn exposure on bone matrix formation assay using murine fetal half calvaria. Neonatal half calvaria was cultured for 7 days with basal medium alone (n = 12 per group) or with 20 μmol/L ZnSO4 (n = 6 per group) or with 100 nmol/L corticosterone (CS, n = 6 per group). Bone area was measured in three images per section. New bone area was calculated by subtracting the original bone area from the total bone and shown as the percentage of the total bone. Blank bar, pups from N-diet-fed dams; striped bar, pups from L-diet-fed dams. Data are presented as means ± SEM. Results for two-way ANOVA within the four groups except for two CS groups are as follows: P = 0.0018 as dam effect, P = 0.2712 as Zn addition effect, and P = 0.6178 as interaction effect between dam and Zn addition

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Mineral Status of Dams

Notably, low Zn concentration was seen in the serum for the L-diet-fed dam group. The dams that were fed the L diet exhibited significantly lower Zn content in femurs compared with those that were fed the N diet. In contrast, serum concentration and femur content of phosphorus and calcium did not change between the dietary groups (Table 4).

Table 4 The Effects of Maternal Dietary Zinc Levels on Mineral Content of Dams
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Serum Parameters for Bone Metabolism in Neonates and Dams

A remarkably lower osteocalcin concentration was seen in dam serum in the L-diet group compared with the N-diet group (Table 5). Osteocalcin concentrations of neonatal serum were much lower than those of dam serum and were significantly lower in neonates fed the L diet than those fed the N diet. IGF-I and corticosterone concentrations were not different in the serum for dam groups or in the pooled serum for their littermates.

Table 5 The Effect of Maternal Dietary Zinc Levels on Serum Biochemical Parameters for Bone Metabolism in Dams and Pups
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Discussion

In the present study, we aimed to elucidate the influence of marginal Zn deficiency during prepregnancy and pregnancy periods on bone status of offspring at birth. To our knowledge, the present rodent model study demonstrated three novel findings: (1) the impairment of neonatal bone matrix formation associated with marginal Zn deficiency during pregnancy, (2) the significant influence of Zn and mineral status on neonatal bone, and (3) the significant reduction of serum osteocalcin concentrations in marginal Zn-deficient dams and their offspring.

In the present study, we set a dietary Zn level at 7 mg/kg as marginally Zn deficient during prepregnancy and pregnancy, compared with 35 mg/kg as control level of dietary Zn in the present study. Human populations are more likely to consume diets that are only marginally deficient in Zn. Additionally, low-Zn diets are started at conception in most animal studies, whereas, in practice, women eating low-Zn diets have probably consumed such a diet prior to conception. Severe depletion of Zn can evoke anorexia in experimental animals [16, 17], causing them to develop marked calorie and protein deficiencies. Anorexia associated with low-Zn diets was not observed in the present study because the Zn concentration of 7 mg/kg, although insufficient, was high enough to prevent anorexia. Therefore, the study of the effects of a moderately low-Zn diet is more relevant for understanding marginal Zn deficiency in human than studies using extremely low Zn-level diets.

We cultured neonatal calvaria to examine the effects of neonatal Zn deficiency on bone matrix formation activity. Calvarial collagen fiber formation, which we designated as the parameter of bone matrix formation, was impaired in the neonates from marginally Zn-deficient dams. The impaired bone matrix formation is probably associated with a reduction of osteoblastic activity because the bone matrix formation process in the calvaria mainly consists of intramembranous ossification by osteoblasts [18]. A previous in vitro study using a primary culture of calvaria showed that Zn significantly induced collagen formation [8]. A chick study also demonstrated that Zn deficiency decreased bone collagen turnover [8]. These findings support the results of our study showing a reduction of neonatal calvaria formation with Zn deficiency in dams. On the other hand, the impairment of bone matrix formation could not be recovered even with the addition of Zn to the culture medium to bring it to the normal Zn concentration present in adult rat blood. This suggests that some critical programming effect on bone matrix formation might be evoked during the period of pregnancy with Zn deficiency. According to a recent report, marginal Zn deficiency during fetal life induced an increase in arterial blood pressure; such prenatal influence could not be totally reversed by postnatal Zn repletion [19]. A long-term study would shed light on the hypothetical programming effect of marginal Zn deficiency during fetal life on bone status in later adult life.

We first demonstrated that femoral phosphorus content was significantly decreased in the offspring of dams fed a marginally Zn-deficient diet, and a similar tendency was observed in femoral calcium content. A previous study in the rhesus monkey [12] revealed the impairment of endochondral ossification in the long bone of offspring with marginal Zn deficiency from conception to the postnatal period. We speculate that bone mineralization in the neonatal femur at birth might be impaired by marginal Zn deficiency of dams during pregnancy. Neonatal long-bone matrix formation is mainly the result of endochondral ossification by chondrocytes [18]. On the contrary, phosphorus and calcium content of calvariae in neonates did not differ between the groups in spite of impaired calvarial bone matrix formation in the neonates from marginally Zn-deficient dams.

These results suggest that bone matrix formation and bone mineralization in long bones might be more susceptible to Zn deficiency compared to calvaria.

Serum osteocalcin concentration, as a bone matrix formation parameter, was evaluated in our study to clarify the bone status of neonates from dams with marginal Zn deficiency. We observed significant reductions of serum osteocalcin concentration in dams and their offspring in the marginally Zn-deficient group. Decrease in serum osteocalcin concentration has been reported in deficient models with less than 1-ppm Zn diet [20]. In the skeletal system, osteocalcin is secreted by osteoblasts and chondrocytes [18]. Taken together, we suggest that neonatal bone matrix formation at birth might be impaired through the compromise of fetal osteoblasts and chondrocytes by prolonged marginal Zn deficiency, including the pregnancy period.

We demonstrated that marginal Zn deficiency during pregnancy resulted in the reduction of Zn content of carcasses and bones in the offspring and in bone Zn content of dams at delivery. The flux of Zn from dams to their rapidly growing fetuses is reported to be sustained at the expense of maternal hepatic pools and other slow turnover tissues, such as bone, when dietary Zn is marginal [23]. According to a previous report using a tracer technique, pregnant rats mobilize Zn from metabolically active pools for transfer to the fetus [22]. Nevertheless, in the neonate, the average ratio of Zn content in the deficient group to that of the control group tended to be lower in bones (70.8% for femur and 71.3% for calvaria) than in carcasses (81.2%). We assume that Zn is preferentially transferred from dam to fetus in tissues with high Zn demand other than bones, which may imply that bones are more susceptible to Zn deficiency than other fetal tissues. Although relatively little information is available on Zn toxicity in human [23], high exposure to Zn and a diet rich in Zn may also adversely affect pregnancy outcome. Therefore, the appropriate intake of Zn is necessary during pregnancy in view of Zn transfer from mother to fetus.

In conclusion, marginal Zn deficiency during pregnancy decreases bone matrix formation activity and bone mineral content in neonates. We strongly propose the appropriate intake of Zn during pregnancy for the proper development and growth of offspring.