Rats born to mothers fed a zinc-deficient diet during pregnancy and lactation displayed stunted growth and decreased expression of the ZnT-2 zinc transporter in the small intestine compared to rats born to mothers fed an adequate zinc diet. Offspring of severely zinc-deficient mothers died or had spinal defects. The downregulation of the ZnT-2 transporter, which exports zinc out of cells, helps maintain zinc homeostasis in conditions of deficiency by reducing zinc efflux. The results emphasize the importance of adequate maternal zinc intake to prevent adverse outcomes in offspring such as stunted growth and mortality.

1. Decreased ZnT-2 transporter expression in offspring produced by
zinc-deficient rats
Aroldo M. Trejo
ABSTRACT
Background: Zinc deficiency during pregnancy has been associated with detrimental outcomes
including infant stunting and spinal defects. ZnT-2 transporters facilitate the efflux of zinc out of
cells and have been shown to alter in expression with dietary zinc intake.
Objective: The objective of the study was to determine how maternal zinc deficiency affects the
growth rate of the offspring and to determine how ZnT-2 expression changes to maintain
homeostatic control in the pups.
Design: Female rats were fed either a zinc adequate (25mg/kg), zinc deficient (7mg/kg) or
severe zinc deficient (1mg/kg) diet for 21 days. Female rats were then mated, they maintained
their respective diets throughout pregnancy and lactation. Offspring were weighed three times
prior to execution and RNA extraction from the small intestine. ZnT-2 gene expression was
analyzed via reverse transcription-polymerase chain reaction and gel electrophoresis.
Results: Offspring of mothers fed a zinc deficient diet displayed stunted growth compared to
those fed an adequate zinc diet. Severe zinc deficient mothers produced deceased offspring with
spinal defects. ZnT-2 transporter expression was decreased in the offspring of zinc-deficient
mothers.
Conclusion: The results from our study emphasize the crucial need to monitor and implement
zinc supplementation in populations with a high prevalence of deficiency in order prevent infant
stunting and mortality caused by maternal zinc deficiency.

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INTRODUCTION
Zinc is crucial in maintaining proper physiological functioning; it plays an important role
in metalloenzyme formation and regulating protein production (1). The movement of zinc across
cellular membranes is regulated by two families of zinc transporters, ZnT (SLC30) and ZIP
(SLC 39) transporters (2). ZnT transporters allow for the influx of zinc from the extracellular
matrix, while ZIP transporters regulate the efflux of zinc from the intracellular cytoplasm (2).
These transporters have been an area of interest, considering their possible role in maintaining
zinc homeostasis, however the exact mechanism of their actions has yet to be elucidated (3, 4).
Expression of ZIP and ZnT transporters has been previously studied by extracting RNA
from the small intestines of rats exposed to various zinc diets (3-5). Reverse transcription and
polymerase chain reaction (RT-PCR) has been utilized to synthesize DNA from the extracted
RNA, amplify the desired genetic sequence using specific engineered primers, and separating the
DNA using gel electrophoresis, ultimately allowing for visualization of transporter expression
(4).
Zinc deficiency during pregnancy has been associated with infant stunting, skin lesions,
and spinal cord defects, and death in severe cases (1, 6-9). Previous studies have examined the
expression of ZnT-2 transporters in order to elucidate their expression in rats exposed to zinc
deficient diets (3), however the link between maternal zinc status and ZnT-2 expression in
offspring has not been studied extensively. The objective of our study is to provide evidence on
the how parental zinc intake influences the expression of ZnT-2 transporters in the offspring and
how this affects their growth rate. We hypothesized that rats born to zinc deficient mothers
would display decreased ZnT-2 expression compared to rats fed an adequate zinc diet. We also

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hypothesized that offspring of mothers fed a zinc deficient diet would yield decreased growth
rates compared to the control rats.
METHODS
Zinc Diet Formulation and RNA Extraction
Female Sprague-Dawley rats were fed either a zinc adequate (25mg/kg), zinc deficient
(7mg/kg) or a severe zinc deficient (1mg/kg) diet for 21 days, followed by a conception period
with wild-type male rats. Female rats maintained the diet throughout conception, pregnancy, and
lactation. Diets were analyzed with atomic absorption spectrometry for zinc level consistency.
Rat litters from the three treatments were weighed at birth, day 14, and day 21. RNA was
extracted from the small intestine of the pups on day 21 using the TRIzol method as described in
previous protocols (5).
RT-PCR and Gel Electrophoresis
Single stranded DNA was synthesized from isolated RNA using High Capacity cDNA
Reverse Transcription Kits by Applied Biosystems (2.0uL 10x RT buffer, 0.8 uL 25x dNTP
100mM mix, 2.0 uL 10x RT random primers, 1.0uL MultiScribe reverse transcriptase, 1.0uL
RNase inhibitor, 3.2 uL nuclease-free H20). 2.0ug of RNA from zinc adequate control pups
(n=12) and zinc inadequate pups (n=12) were added, the resulting mixture was centrifuged and
heated in a Thermal cycler as follows: 25°C for 10.0 minutes, 37° for 120.0 minutes, and 85° for
5.0 minutes. Five samples did not utilize the RNase inhibitor, 1.0uL of nuclease-free H20 was
substituted to maintain 20uL volume.
cDNA samples were amplified using Thermo Scientific Phusion High-Fidelity PCR Kits
(11.6µl H2O, 4.0ul 5x Phusion HF buffer, 0.4ul 10mM dNTPs, 1.0ul ZnT-2 reverse primer, 1.0ul
ZnT-2 forward primer, 1.0ul template DNA, 1.0ul working Phusion DNA Polymerase). The

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samples were heated in the Thermal cycler for 35 cycles as follows: 98°C 1:05 minutes., 58°C
for 0:15 minutes., 72°C for 2:30 minutes., ultimately concluded with a final extension period at
72°C for 10 minutes.
Gel electrophoresis was conducted on 1% agarose gel and 1xTE buffer for 45.0 minutes
at 90V. The twelve samples were separated into two rows on the agarose gel and measured using
8.0cm GeneRuler 100bp DNA ladders. Gene expression was visualized using
RESULTS
Dietary Effect on Growth
Average growth rates of the pups born from zinc adequate and inadequate mothers is
depicted in Figure 1. Mother rats consuming an adequate zinc diet produced offspring with
greater growth compared to rats fed a deficient zinc diet. At day 21, pups from the mothers
consuming a zinc adequate diet were 39% larger than their counterparts. Mother rats consuming
a severe deficient zinc diet (1mg/kg) yielded rats that died at before day 14 measurements, thus
their data was excluded from the comparison.
Figure 1. Rat pup size comparing zinc adequate (left) and zinc deficient (right) conditions at birth (A) and
21 days (B). Severe zinc deficient diets resulted in myelomeningocele and pup death (C).

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Figure 2. Average rat pup growth between mothers fed an adequate zinc diet and rats fed a zinc deficient
diet. Mother rats fed an adequate zinc diet yielded greater rat pup growth compared to zinc deficient rats
at all measurement days.
RT-PCR Electrophoresis
Results from RT-PCR electrophoresis indicating the gene expression of the ZNT-2
transporters in both conditions is displayed in Figure 3. All samples on the agarose gel ran
below the lowest rung on the DNA ladder, indicating a gene size of less than 200 bp. Due to
limited space on the agarose gel, the size of gene sequence could not be adequately quantified.
However, in all samples, the rats fed an adequate zinc diet displayed expression of the ZNT-2
transporter, while those fed zinc deficient diet yielded a muted response. This is evident in
samples C, D, G, K, and L, where expression of the ZNT-2 transporter in the adequate zinc diet
was highly expressed.
Day
1
Day
14
Day
21
Zinc
Adequate
(25mg/kg)
(g)
6.8
23
32
Zinc
Deficient
(7mg/kg)
(g)
4.5
12.5
23
0
5
10
15
20
25
30
35
Weight
(grams)
Zinc
Status
Effect
on
Rat
Growth
Rate

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Figure 3. RT-PCR gel electrophoresis displaying gene expression of the ZNT-2 transporter from rats fed
an adequate zinc diet (C) or a zinc deficient diet (I). * indicates the use of an RNAse inhibitor.
Samples A-E, H, and K utilized an RNAse inhibitor during the reverse transcription
process. Samples that used the RNAse inhibitor did not display enhanced gene expression
compared to samples that did not utilize the RNAse inhibitor. Samples E, F, I, and J yielded
decreased gene expression compared to the other samples.
DISCUSSION
RT-PCR and Gel Electrophoresis
RT-PCR allowed for isolation, amplification, and visualization of ZnT-2 gene expression
via RNA isolated from the small intestines of the pups. Gel electrophoresis of ZnT-2 transporter
DNA yielded diminished expression in the pups born from mothers consuming a zinc deficient
diet (Figure 3). It is interesting to note that samples E, F, I, and J yielded decreased gene
expression compared to the other samples. This may have been due to experimental error in the
PCR process; key components may have been excluded due to the small quantities utilized in
this experiment, such as the addition of polymerases or primers. The lack of these components

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may have resulted in stunted DNA synthesis and replication, subsequently decreasing the
visualization of ZnT-2 transporters in these samples.
All samples ran below the lowest rung on the DNA ladder (200bp), resulting in
inadequate measurements of the ZnT-2 gene. Limited space on the agarose gel resulted in the use
of two 8.0cm DNA ladders to separate the twelve samples, not allocating enough room for the
use of a larger ladder. A larger ladder would have allowed for greater accuracy in the
quantification of the ZnT-2 gene analyzed. Previous studies have quantified ZnT-2 transporters
derived from rat intestines to be between 100bp and 200bp, which falls relatively in line with the
results from our study (4).
No significant difference was found between samples that used the RNase inhibitor in the
reverse transcription process. RNase degrades the RNA samples at an optimal temperature of
60°C (10), resulting in decreased expression following RT-PCR and gel electrophoresis. All
samples were iced immediately upon addition of the components throughout the RT-PCR
process, minimizing the effects of RNase and possibly depicting the lack of RNase inhibitor
efficiency.
ZnT-2 & ZIP Transporters Mediation in Zinc Homeostasis
ZnT-2 was found to be down regulated in the offspring of the mother rats consuming a
zinc deficient diet, supporting our initial hypothesis and depicting the importance of the ZnT-2
transporter in maintaining zinc homeostasis. The ZnT family of transporters function to facilitate
the efflux of zinc from the intracellular space of cells to the extracellular matrix (2). A study
conducted by Liuzzi et al. studied the expression of rat ZnT transporters in response to a two
week feeding period of a zinc deficient (less than 1mg/kg), adequate zinc (30mg/kg), and
supplemental zinc (180mg/kg) diet (3). The study yielded no expression of ZnT-2 transporters in

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the intestines of the rats fed a zinc deficient diet compared to the rats fed an adequate zinc diet;
rats fed supplemental zinc displayed a significant increase in the expression of ZnT-2
transporters compared to the control (P<0.05). A study conducted by Pfaffl and Windisch found
a similar significant decrease in ZnT-2 transporter expression in the jejunum of mice fed a zinc
deficient diet (p=0.098) (4). The results from these studies concluded that the expression of ZnT-
2 transporters correlated with fluctuations in the dietary zinc status of the mice (3, 4). The results
from our study correlate with these results, and add further evidence that a deficiency in zinc has
lasting heritable effects on ZnT-2 expression, as depicted by the decreased expression of the
transporters in the pups born to zinc deficient mothers.
The decrease in ZnT-2 transporters correlates with an attempt to maintain zinc
homeostasis in response to dietary zinc changes. Reduced ZnT-2 transporters decrease the efflux
of zinc out of cells, providing a protective mechanism in light of zinc shortages (3).
Alternatively, previous studies have found an increase in various ZIP zinc transporters in
response to decreased zinc intake (5, 11), although the magnitude of the response is tissue
dependent. The ZIP zinc transporters are membrane proteins that act in an opposite manner of
the ZnT family and promote the influx of zinc from the extracellular matrix into the cell or
organelle (2). Ultimately, increases in various ZIP transporters and decreases in ZnT zinc
transporters function to maintain zinc levels in mice, displaying a tight regulation of zinc
homeostasis (12). Our study did not analyze the expression of ZIP transporters, however the loss
of expression of ZnT-2 transporters in pups born to zinc deficient mothers confirm a decrease in
the efflux of zinc, exemplifying a homeostatic response to retain zinc.

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Consequences of Zinc Deficiency
The results from our study demonstrated the heritable morphological consequences of a
zinc deficient diet, resulting in growth-stunted pups in deficient conditions and deceased rats in
severely restricted conditions (Figure 1). Zinc plays an important role in energy and protein
metabolism; rat studies have confirmed that a lack of zinc down regulates the production of
important enzymes that ultimately results in growth retardation and spinal defects (9). These
results have been consistent in human studies, depicting the importance of an adequate zinc diet
during pregnancy in ensuring prenatal health (8).
Additionally, recent human studies have found an association between decreased ZnT-2
transporters in mammary cells and lowered zinc concentration in breast milk, ultimately resulting
in a zinc deficiency in newborns (13, 14). A mutation in the ZnT-2 gene results in a lack of
transporters in mammary tissue, resulting in impaired zinc exportation in breast milk (13, 14).
Breast milk is an important source of zinc for newborns; requirements for zinc increase in order
to facilitate rapid growth in infants (15). Our study depicted stunting in pups born to zinc
deficient mothers; mothers were fed a zinc deficient diet throughout pregnancy and lactation.
Although our study did not quantify ZnT-2 transporters in parental rats, results from previous
studies allow us to conclude probable decreased ZnT-2 transporters in zinc deficient mothers (3).
This may have decreased the amount of ZnT-2 transporters in mammary glands, decreasing the
zinc content in breast milk and further inducing stunted growth in the pups. More research needs
to be conducted in order to gain validity in this claim, nonetheless the results of our study
implicate the crucial need to monitor maternal zinc status throughout pregnancy in order to
ensure proper infant growth.

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The effects of maternal zinc deficiency have been shown to be reversed with early
supplementation; recent studies have shown an increase in infant weight upon initiation of
maternal zinc supplementation, depicting the importance of zinc monitoring in pregnant mothers
in order to ensure prenatal health (6, 7). Considering the manageability of zinc deficiency via
early supplementation, the results from our study emphasize the importance of proper screening
for zinc deficiency in order to prevent infant stunting and death.
CONCLUSION
Our study revealed decreased ZnT-2 transporter expression in the offspring of mother rats
fed a zinc deficient diet, supporting our initial hypothesis and depicting the tight regulation of
zinc in order to maximize its retention. Furthermore, maternal zinc deficiency induced in our
study resulted in stunted pup growth, while severe maternal zinc deficiency resulted in spinal
defects and pup death. The results from our study emphasize the crucial need to monitor and
implement zinc supplementation in populations with a high prevalence of deficiency in order
prevent infant stunting and mortality. A weakness of our study included the lack of analysis of
ZIP transporter gene expression in the offspring, our analysis focused primarily on the efflux of
zinc rather than the combined activity of both transporter families. This study has propagated
future studies in zinc supplementation, in order to further solidify whether infant
supplementation can reverse parental zinc-deficient induced stunting.
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