BIOLOGY OF REPRODUCTION (2016) 94(4):86, 1–14

Published online before print 10 February 2016.

DOI 10.1095/biolreprod.115.137240

Minireview

Trace Elements in Ovaries: Measurement and Physiology1

Melanie J. Ceko,3 Sean O’Leary,4 Hugh H. Harris,3 Katja Hummitzsch,4 and Raymond J. Rodgers2,4
3
Department of Chemistry, The University of Adelaide, South Australia, Australia

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4
Discipline of Obstetrics and Gynaecology, School of Medicine, Robinson Research Institute, The University of
Adelaide, South Australia, Australia

ABSTRACT zinc (Zn), cobalt, fluorine, iodine, manganese, molybdenum,

nickel, silicon, and vanadium, with some disagreement over
Traditionally, research in the field of trace element biology arsenic, chromium, and tin being essential trace elements [2–4].
and human and animal health has largely depended on Evidence for essentiality of a particular trace element is
epidemiological methods to demonstrate involvement in biolog-
established when symptoms are induced by dietary deficiency,
ical processes. These studies were typically followed by trace
element supplementation trials or attempts at identification of
then subsequently reversed after a diet that is replete in this

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the biochemical pathways involved. With the discovery of
specific trace element is provided [5]. Essentiality is then
biological molecules that contain the trace elements, such as generally acknowledged when it has been demonstrated by
matrix metalloproteinases containing zinc (Zn), cytochrome more than one independent investigator and in more than one
P450 enzymes containing iron (Fe), and selenoproteins contain- animal species [6]. Evolutionary selection of these elements
ing selenium (Se), much of the current research focuses on these remains a mystery; the property that the suite of elements
molecules, and, hence, only indirectly on trace elements discussed in this review has in common is that they commonly
themselves. This review focuses largely on two synchrotron- occur and function in living organisms at low concentrations.
based x-ray techniques: X-ray absorption spectroscopy and x-ray Interest in the potential role of trace elements in human and
fluorescence imaging that can be used to identify the in situ animal physiology began over a century ago with the discovery
speciation and distribution of trace elements in tissues, using our that a number of compounds in living organisms contained
recent studies of bovine ovaries, where the distribution of Fe, Se, metals not previously considered to be of biological signifi-
Zn, and bromine were determined. It also discusses the value of cance [6]. When studying the relationship of trace elements in
other techniques, such as inductively coupled plasma mass human health, it becomes increasingly evident that optimal
spectrometry, used to garner information about the concentra- levels of elements in every organ, tissue, and cell of the human
tions and elemental state of the trace elements. These

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body may be key to maintaining a healthy existence [7]. Trace
applications to measure trace elemental distributions in bovine elements are present in every biological process, from the
ovaries at high resolutions provide new insights into possible production of hormones and energy, digestion, nerve trans-
roles for trace elements in the ovary. mission, and muscle contraction to the regulation of pH,
bromine, iron, ovary, selenium, synchrotron x-ray fluorescence, metabolism, and cholesterol and blood sugar levels [7]. Stress
trace elements, zinc and exposure to environmental pollution alter our requirements
for these minerals, and scientists are becoming increasingly
INTRODUCTION aware that trace element deficiencies can affect both physical
and mental health.
When living organisms migrated to the land from water In addition to identifying trace elements, elucidating the
during the Paleozoic period, they had to depend on the soil for roles they might be playing at a biochemical level, and
their source of biological trace elements [1]. A total of 26 of the considering the optimal dietary intake, bioavailability, or
90 naturally occurring elements are known to be essential for amount of utilizable element relative to the amount present in
animal life, including copper (Cu), iron (Fe), selenium (Se), the daily diet, are important considerations [8]. Trace element
1
bioavailability is influenced by a complex matrix of interacting
Funding was received from Australian Research Council grants variables, including the chemical form of the element found in
DP0985807 and DP0984722 and the National Health and Medical
Research Council of Australia.
food, the nature of the food ingested, the composition of the
2
Correspondence: Ray Rodgers, School of Medicine, Robinson Re- total diet, and the health and nutritional well being of the
search Institute, The University of Adelaide, SA 5005, Australia. individual [3, 9]. Dietary deficiencies of trace elements have
E-mail: ray.rodgers@adelaide.edu.au been reported to alter various aspects of reproductive
physiology [10]; however, to date, the majority of these
Received: 20 November 2015. studies have focused on their roles in male reproductive
First decision: 17 December 2015. function.
Accepted: 28 January 2016. Traditionally, research in the field of trace element biology
Ó 2016 by the Society for the Study of Reproduction, Inc. This article is and human health has largely depended on epidemiological
available under a Creative Commons License 4.0 (Attribution-Non-
Commercial), as described at http://creativecommons.org/licenses/by-nc/
methods to demonstrate involvement in biological processes.
4.0 These studies were typically followed by trace element
eISSN: 1529-7268 http://www.biolreprod.org supplementation trials or attempts at identification of the
ISSN: 0006-3363 biochemical pathways involved. With the discovery of
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 CEKO ET AL.

Relative cost of analysis takes into account preparation of sample, start-up costs, etc. S-XRF and ICP-MS require expensive equipment that is normally shared between institutions, allowing for assay costs to
Relative costc
biological molecules that contain the trace elements, such as

Medium

Medium
matrix metalloproteinases containing Zn, superoxide dismutase
Low

Low
containing Mn or Cu, cytochrome P450 enzymes containing
Fe, or selenoproteins containing Se, much of the current
research focuses on these proteins and, hence, only indirectly
on trace elements themselves. While useful, these approaches
overlook key roles of many trace elements in biological

rather than elemental

Low, generally used to processes, including reproduction. In this review, we cover
provide chemical some of the newer techniques for studying trace elements and,
Accuracyb

in particular, their application to the study of the ovary.

Medium to high
(quantitative)
information
NEWER TECHNICAL APPROACHES TO STUDY TRACE
Medium

ELEMENTS
High

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The objectives of newer approaches to study trace elements
in cells and tissues are first to identify which trace elements are
present, to quantify levels of trace elements, and to determine
the location of the trace elements in tissues and cells.
Accuracy: nearness of result to the true result, is dependent on sample preparation, homogeneity of tissue, and interfering matrix effects (see text).
U), nonsimultaneous

According to the findings of the study of these parameters,

Range of elementsa

Multielement (Mg to

Multielement (Mg to

assumptions can be made with regard to tissue- and cell-

U), difficult with
lighter elements

Range of elements is generally considered to be from Mg to U for XAS and XRF, and lighter elements are possible with S-XRF and ICP-MS.

specific function of trace elements. In a paper designed to

simultaneous

simultaneous

evaluate laboratory methods for trace element determination,

Multielement,

Multielement,

Bolann et al. [11] summarized that the analysis of trace

elements in biological fluids and tissues serves multiple

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purposes. These include determining the concentration and
be relatively low (shared) compared with platforms using less-expensive equipment, but operated within individual institutions.

distribution of essential trace elements in normal and disease

conditions, detection and allocation of potentially toxic metals,
diagnosis of trace element deficiency states, and trace element-
related diseases [11]. According to their findings, the four most
Yes, morphology intact.

Possible, although bulk

In situ quantification

commonly used techniques for trace element analysis in human

biological material are: 1) flame atomic absorption spectrom-
Single-cell level

analysis more

etry, 2) graphite furnace atomic absorption spectrometry, 3)

inductively coupled plasma atomic emission spectrometry, and
common

4) inductively coupled plasma mass spectrometry (ICP-MS),

with ICP-MS being preferable for simultaneous screening of
multiple elements [11].
No

No

In another study designed to ascertain the most effective

means of analyzing the elemental composition of a geological

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sample (samples of sediment from the bottom of a lake),
Phedorin et al. [12] compared synchrotron x-ray fluorescence
powder or emulsion
No preconditioning of
Tissue preparation

(S-XRF), traditional XRF, ICP-MS, atomic absorption spec-

Decomposition to

Decomposition to

trometry, and instrumental neutron activation analysis, and

TABLE 1. Newer technical approaches to study trace elements in tissues.

concluded that S-XRF was superior from the point of view of

liquid phase
Unnecessary

its speed, ease of application, cost, nondestructive nature, and

sensitivity [12]. These factors, coupled with the simultaneous
tissue

determination of multiple elements of geochemical or bio-

chemical interest, make synchrotron-based XRF a highly
sought-after technique.
Weekley et al. [13] assert that XRF imaging and x-ray
absorption spectroscopy (XAS) are ideal techniques for
0.02–0.5 g (dry weight)

investigating the chemical speciation and distribution of

1 mg–1 g (wet weight)

0.2 mg (dry weight)

elements heavier than silicon in biological systems, with

Mass of tissue

1–7 g (dry weight)

minimal sample preparation required [13]. XRF imaging is

now a well-established, nondestructive analytical method
applicable across a large variety of fields, including materials
science, environmental science, geology, life science, and
archeological science [14]. The discussion below describes the
newer approaches to studying trace elements, detailing the
basic principle behind the approach, including the advantages
and limitations of each platform. Table 1 compares the
platforms with regard to sample preparation and salient
Analytical platform

features.

S-XRF Imaging
ICP-MS
S-XRF

The principle of XRF involves using x-rays of sufficient

XAS
XRF

energy to expel tightly held inner electrons of an element

b
a

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FIG. 1. Schematic diagram demonstrating the physical principles associated with S-XRF. 1) X-rays that strike an atom may knock electrons out of it,
leaving behind an ionized version of the atom (i.e., one missing electron), and an unoccupied electron energy level. 2) An electron of a higher energy will
then drop down to fill the lower-energy, unoccupied level, releasing an x-ray photon as a means to conserve energy. 3) The emission of the x-ray photon is
the source of the fluorescence. The electron energy levels are very specific to each element, so that the energy of the S-XRF produced precisely describes
the elements present in the biological sample.

within a sample. This makes the electronic structure of the Applications of S-XRF Imaging to Biological Systems
element unstable, and an electron from a higher orbit replaces
the inner electron, at the same time releasing energy in the form To date, there is a plethora of studies, ranging from

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of a fluorescent photon. The fluorescent photon has a elemental tissue profiles and identifying toxic metals in human
characteristic energy equal to the difference in energy between teeth [19–21], to determining levels of calcium and phospho-
the outer and inner orbital [15]. As the electron energy levels rous in the bones in osteoporosis research [22, 23], and
are specific for each element, the fluorescence produced by the comparing chemical composition in liver biopsies from
x-ray precisely identifies the elements within the sample (see untreated cirrhosis patients with healthy controls [19, 24].
Fig. 1). However, only a limited number of XRF imaging studies have
A synchrotron is a circular particle accelerator that uses both been carried out involving male or female reproductive organs.
magnetic and electric fields to accelerate charged particles. Quantitative analysis of Zn and Ca in human prostate cancer
During this process, bunches of electrons are accelerated to and normal tissues by S-XRF imaging was undertaken by Ide-
about the speed of light within an orbital storage ring, and give Ektessabi et al. [25] in order to investigate differences in the
off synchrotron radiation, which can then be harnessed for the distribution and concentrations of Zn in these tissues. Ortega et
x-ray source for XRF (see Fig. 2). S-XRF imaging is a al. [26] mapped the distribution of chromium in the
powerful technique that harnesses the finely focused x-ray reproductive organs of male mice after exposure to CrCl3 to
beam that is several orders higher in intensity than radiation elucidate the specific cellular regions where it had accumulat-
from traditional x-ray sources, and focuses this onto a small ed. Two more recent papers presented S-XRF imaging of the
spot on a biological sample (e.g., an ovarian section). Digital ovaries to investigate the bioaccumulation of dietary Zn in
images of microscopic tissue samples are built, pixel by pixel, Daphnia magna, a common ecotoxicological model [27, 28].
by scanning the sample through the beam, and mathematical In both cases, the researchers set out to investigate the
deconvolution analysis of the fluorescence spectrum reveals the hypothesis that Zn selectively accumulates in female repro-
elemental composition. Quantitative elemental distribution ductive tissues and adversely effects reproduction. S-XRF has
maps of the sample can be assembled [16] to reveal the spatial also been used in recent years to analyze the trace elemental
organization of trace elements within the sample with high composition of human breast tissue, and has revealed
spatial resolution and sensitivity (to ppm or better) for a large statistically significant elevation of a multitude of elements in
number of elements (Fig. 2) [17]. The S-XRF process is
neoplastic tissue compared to normal samples [29–33].
undertaken without destroying the tissue under study [18].
In summary, the use of S-XRF in elucidating the
distribution and quantification of both essential and toxic trace
elements in tissues provides new insights into diseases and
provides ever-increasing applications to biological systems
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FIG. 2. Schematic representation of S-XRF. The sequence of steps includes: 1) X-rays produced by highly energetic electrons accelerated in the
synchrotron storage ring; 2) x-ray optics are then used to energy filter; and 3) focus synchrotron sourced x-rays onto a small spot (several nanometers to
micrometers in diameter) on the sample; 4) the sample is then raster scanned through the beam; 5) an energy-dispersive fluorescence detector is used to
produce a full-fluorescence spectrum; and 6) peaks correspond to specific elements. By comparing these spectra to reference standards of known
concentration, quantitative in situ maps of multiple elements (7) can be created simultaneously.

[34]. However, despite there being about 90 synchrotrons useful in ascertaining which biological molecules the elements

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worldwide, only a handful have the capability of XRF imaging, are associated and, hence, their functions.
and even fewer have the advanced capability to collect large
amounts of data quickly, as was recently undertaken in the Inductively Coupled Plasma-Mass Spectrometry
ovary [35, 36].
The principle of ICP-MS involves a high-temperature
inductively coupled plasma source, which converts the atoms
X-Ray Absorption Spectroscopy of the elements within a sample into gaseous ions. These ions
The principle of XAS is similar to XRF, where synchrotron- are then separated and detected by mass spectroscopy due to
sourced x-rays are used to ionize core electrons, creating an their mass-to-charge ratio. The multielemental capability of
absorption edge with an energy that is characteristic to each ICP-MS for the analysis of biological samples makes this an
element. XAS determines the local geometric and/or electronic ideal supplementary technique for quantifying the trace
structure of trace elements in matter [37]. More specifically, elements identified by XRF imaging. ICP-MS is capable of
information can be gleaned on bond lengths, coordination detecting metals and several nonmetals at concentrations as
numbers, local coordination geometry, and the oxidation state low as one part per trillion [41].
of atoms for a wide range of solid or liquid samples.
Traditionally, the XAS spectrum is divided into two regions: ROLE OF TRACE ELEMENTS IN FEMALE REPRODUCTIVE
the low-energy region, which covers photon energy up to about FUNCTION
50 eV above the absorption edge, often referred to as x-ray Over the last four decades, countless studies have been
absorption near-edge structure (XANES); and the higher- conducted on the role of dietary trace elements on reproductive
energy region, from 50 to 1000 eV above the absorption edge, function; however, a review of the literature quickly highlights
which is called the extended x-ray absorption fine structure how heavily the research has been weighted toward male
(EXAFS) [38, 39]. Although the two regions have the same compared to female reproductive function. Studies with a great
physical origin, this distinction is convenient for their deal of evidence on the impact of micronutrients, including
interpretation [40]. XANES is highly sensitive to the spectral trace elements, on female fertility are rare [42]. Although there
oxidation state and coordination chemistry of the absorbing are many human and animal studies focusing on the dietary
atom, while the EXAFS is used to determine the distances, intake of trace elements during pregnancy, and the resultant
coordination number, and identity of the neighboring atoms. effects on pregnancy outcome, very few studies take into
Thus, the information obtained using XAS is biologically account the preconception period, or focus on general
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TABLE 2. Trace elements and proposed effect on ovarian function.

Element Bromine Iron Selenium Zinc

Source Soils, anthropogenic Food/supplements Food/supplements Food/supplements

release
Proposed mechanism/ Halogen replacement Cell growth/function, #ROS/oxidative stress #ROS/oxidative
function BDBP & PBBs, collagen LH & FSH secretion stress
formation
Deficiency # Tissue integrity[56] Infertility [102], Unexplained infertility # Oocyte maturation
anovulation [193], [194], POF (?) anovulation [195]
hypoxia (?)
Toxicity Transgenerational infertility Infertility [103, 104] n/a n/a
[78] iron-induced
oxidative stress (?)

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BDBP, brominated disinfection by-product; n/a: information not available; POF, premature ovarian failure; (?), putative mechanism.

reproductive health when it comes to ovarian function, Br-deficient goats showed abnormalities in tissue sections of
including hormone synthesis and follicular development. the thyroid, heart, lungs, pancreas, and ovaries [51]. In 1998, it
In the late 1990s, Xu et al. hypothesized that a disorder in was suggested that, although these additional studies gave
the metabolism of trace elements may be one of the important more credibility to the concept of Br essentiality, the findings
factors causing unexplained infertility [43]. Recent studies, were still regarded as too limited [52]. The evidence provided
which are reviewed elsewhere [44], have reported that changes little insight into a possible biochemical role for this element,
in the levels of the trace elements, Fe, Zn, and Ca, play beyond saying that it may serve as an electrolyte [52]. This led
important roles in female infertility, but there is still debate to the general assumption that the biological behavior of Br

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about the relationship between Fe and unexplained infertility was similar to chlorine (Cl) in that the administration of it
[44]. A better understanding of the role of trace elements in the results in some displacement of body Cl, and vice versa [53].
underlying mechanisms in infertility, as well as more rigorous This assumption has since been found to be invalid for the
studies clarifying the effectiveness of nutritional factors, are thyroid gland. In studies on the interaction of Br with I in the
needed to improve diagnosis and treatment [45]. Despite the rat thyroid, under the conditions of enhanced Br intake,
paucity of studies, it is clear that there is a strong relationship Pavelka et al. found that, contrary to other tissues, Br(aq)
between nutritional status of trace elements prior to conception replaced iodide (I) as opposed to Cl [54, 55]. As previously
and successful conception and healthy progression of preg- mentioned, a recent study demonstrated that Br is required as a
nancy [46]. cofactor for peroxidasin-catalyzed formation of sulfilimine
In order to gain a better understanding of the role of trace crosslinks in Drosophila [56]. This process is involved in
elements in the ovary, we recently undertook quantitative XRF cross-linking the noncollagenous domains of collagens type
imaging of bovine ovaries and conducted ICP-MS and XAS IV, which are the collagen components of basal laminas found
analyses [35, 36, 47, 48]. In particular, we obtained data on in many tissues. Br dietary deficiency led to physiologic

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bromine (Br), Fe, Se, and Zn. These trace elements and their dysfunction in Drosophila, while repletion of this trace element
potential roles in the ovary form the basis of this review. reversed the dysfunction, a key criterion for justifying
However, some ovaries showed evidence of other trace essentiality [56].
elements, including Cu, cobalt, chromium, manganese, nickel,
and titanium, but these were not in particularly high Biological Activity of Brominated Species
concentrations, nor were they found in a significant portion
of samples (,10%), and so these elements were not examined Potential biological roles of brominated species have been
further. In the following sections, we discuss what is known under scrutiny for the past 13 yr, with several authors making
about Br, Fe, Se, and Zn in general and in reproduction, and, in important contributions to this area of knowledge. Hawkins et
particular, in the ovary. Table 2 summarizes the proposed al. conducted an experiment to show that hypochlorous acid
effects of these trace elements on female fertility and ovarian (HOCl) and hypobromous acid (HOBr) reacted with different
function. Table 2 also includes proposed mechanisms associ- selectivity with cellular targets, and that the resultant radical
ated with deficiency, and where possible toxicity of these trace formation may result in cell lysis [57]. The reactivity of HOBr
elements is associated with ovarian function. with biological molecules was not well characterized, but
amino acids and proteins appear to be major targets, with the
BROMINE species reacting with fatty acid side chains and lipid-soluble
antioxidants to a much greater extent than HOCl [58–60]. This
Br is one of the most abundant and ubiquitous trace species has also been reported to induce red blood cell lysis
elements in the biosphere, but, until recently, had not been approximately 10 times more rapidly than HOCl [57]. In a later
conclusively shown to perform any essential function in plants, study to ascertain the rate constants for reactions of HOBr with
micro-organisms, or animals [2]. Up until the mid-1980s, only protein components, most residues reacted 30- to 100-fold
weak evidence existed to support the view that Br was faster with this species than with the Cl-containing acid [58].
essential, with one of the key findings being that bromide More recently, the contribution of HOBr to optimal and
(Br[aq]) could substitute for part of the chloride (Cl) efficient microbial killing has been recognized as essential
requirement for chickens [49]. In 1990, one additional study [61]. Marcinkiewicz et al. [62] investigated the role of another
reported that, when compared to goats fed a 20-mg Br/kg diet, brominating oxidant, taurine bromamine, and found that this
goats fed a 0.8-mg Br/kg diet exhibited depressed growth and species exerted strong bactericidal effects on Escherichia coli.
fertility, increased abortions, and reduced life expectancy [50]. Maines et al. [63] investigated the cytotoxic effects of activated
In addition, microscopic examination of tissues and organs of Br on human fetal osteoblasts in vitro and showed that sodium
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tors to the anthropogenic release of Br into the environment

include mining, industrial emissions, disinfectants, flame
retardants, and the use of fertilizers and pesticides in agriculture
[65]. These processes can significantly increase the concentra-
tion of Br in the environment. With such prevalent exposure to
Br, coming from natural and anthropogenic sources, it is not
surprising that numerous studies have been conducted on the
impact of chronic Br exposure on mammalian systems.
Throughout the 1980s, several authors confirmed that large
doses of Br could reduce fertility, impact the rate of survival
of offspring, and result in depressed production of thyroxine,
because excess Br(aq) competes with I in the synthesis of this
hormone [67–69]. Flury et al. [65] suggested that chronic Br

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toxicity affects mainly the endocrine and reproductive systems
of mammals, and Pavelka [64] further suggested that high
Br(aq) levels can influence iodine metabolism by either
decreasing the iodine accumulation in the thyroid and skin
and/or by increasing the excretion of iodine by the kidneys.
More recent studies have focused less on Br(aq) and more on
the potential toxicology of brominated disinfection by-
products. Several authors focusing on dibromoacetic acid have
concluded that chronic exposure to even low levels of this
chemical (1 mg/kg body weight/day) adversely affects

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reproductive function in male rabbits, and delays pubertal
development and compromises sperm quality in rats [70–72].
In humans, most of the research investigating the effects of
Br is centered around the possible toxic effects of brominated
compounds and residues in food and water supplies [73, 74].
As a member of the halogen family, the propensity for
substitution with other halogens (chlorine and iodine in
particular) and the advent of newer technologies that identify
and quantify the bioaccumulation of Br in tissue, more research
is examining the toxic effects of Br exposure on pregnancy
outcome. However, to date, there are inconsistent associations
with spontaneous abortion, preterm birth, small-for-gestational-
age babies, and birth defects [73]. In a study spanning 3
decades, Small et al. [75] investigated the effects of exposure

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of brominated flame retardants and polybrominated biphenyls
(PBBs), a class of halogenated, brominated flame retardants,
FIG. 3. Distribution of Zn, Fe, Se, and Br in bovine ovaries, as imaged by during pregnancy on the fertility and reproductive outcomes of
XRF. Comparison with the adjacent fresh frozen tissue (A) indicates that offspring more than 20 yr after exposure. Of 194 women that
regions of high Zn (B, pink) intensity correspond to arterioles and were exposed to PBBs while in utero, there was a decrease in
capillaries; high Fe (C, pink) correspond to corpora lutea; and high Se (D, fertility and an increased risk of spontaneous abortions with
pink) is localized to the granulosa cell layers of the two largest healthy increasing risk associated with greater exposure to PBBs [75].
follicles. E) H&E-stained section of a bovine ovary containing arterioles The finding of an increase in spontaneous abortions suggests
alongside Br elemental distribution map (F) generated by XRF imaging. 5,
atretic follicles; *, healthy antral follicles; - - - -, corpus luteum or regressed that the in utero exposure on reproductive outcomes is likely
follicles; ", capillaries; ¤, arterioles. Scale bars ¼ 4 mm (A–D) and 200 lm due to direct exposure to the fetal ovaries at the time when
(E and F). Reprinted from Ceko et al. Metallomics 2015; 7:71–82 and early follicular development is initiated. A hypothesis to
Ceko et al. Metallomics, 2015; 7:756–765 [48] and reproduced by account for the findings includes the effect that PBBs have on
permission from The Royal Society of Chemistry. estrogen activity and progesterone production in in vitro
models and animal studies [75]. Concurrent exposure to PBBs
in pregnancy has led to spontaneous abortions in cattle [76,
bromide was more cytotoxic than either sodium hypochlorite or
77], but not in humans [75].
activated sodium hypobromite. Thus, it appears that there is a To our knowledge, our group was the first to identify and
range of chemical modifications induced by Br(aq)-based document the presence and nature of distribution of Br in the
species, many of which are still in the process of being fully mammalian ovary [35]. Qualitative observations of the
elucidated. XANES region of the spectra coupled with principal
component analyses of all XAS data led us to conclude that
Br Byproducts and Reproduction the predominant form of Br was aqueous Br. Using S-XRF,
Br was identified in high concentrations in all sections from 45
In nature, Br is found mostly as Br, bound to metals in the bovine ovaries and was widely distributed throughout the
form of inorganic salts [64]. The main natural source of ovarian tissue, but heavily concentrated in the walls of
Br(aq) is in sea water, where the average concentration is arterioles (Fig. 3F). The only known biological function of
several orders of magnitude larger than in freshwater systems Br is for cross-linking of type IV collagen [56], which is
[65]; however, it is also found naturally in soils [66] and a predominantly a basal lamina component. Cross-linking of
range of plant- and animal-based foods [65]. Major contribu- subunits would presumably increase the tensile strength of
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basal laminas. The basal laminas of subendothelial cells of transferred across the duodenal mucosa into the blood, where
arterioles, venules, and capillaries and the basal laminas of it is transported by transferrin to the cells or the bone marrow
smooth muscle cells of arterioles in bovine ovaries contain type for red blood cell production [94, 95]. Fe uptake, storage, and
IV collagens a1 and a2 [78]. Cross-linking of both type IV export by cells are tightly regulated by Fe regulatory proteins
collagens a1 and a2 has been observed previously in bovine (IRPs) [96]. IRPs are cytosolic trans regulators that bind to
follicles [78]. That Br would be heavily concentrated in the specific RNA Fe-responsive elements that are present within
walls of arterioles would suggest that its function is to facilitate the mRNA encoding transferrin receptors and ferritin, thus
substantial cross-linking of type IV collagens in the subendo- controlling the intracellular flux of Fe [97].
thelial basal laminas and that of the smooth muscles of arterial Dietary Fe occurs in both heme and nonheme forms [98].
walls to withstand the high intra-arteriole blood pressures. Primary sources of heme Fe are hemoglobin and myoglobin
In our study [48], we observed tenfold higher concentrations from consumption of red meat, poultry, and fish, whereas
of Br in bovine serum versus normal human serum concen- nonheme Fe is obtained from cereals, pulses, legumes, fruits,
trations, which raises some important questions regarding the and vegetables [99]. The difference in bioavailability of the two
dietary and environmental exposure to Br for the cattle. Due to

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Fe forms (15%–35% and 2%–20% for heme and non-heme,
previously determined toxic nature of Br, the origin and source respectively), in addition to the fact that dietary factors have
of Br requires further investigation to determine whether it is little effect on absorption of heme Fe, make red meats an
from anthropogenic or natural sources. excellent nutrient source of this trace element [98]. Major
inhibitors of Fe absorption are phytic acid, polyphenols,
IRON calcium, and peptides from partially digested proteins [98].
Fe has the longest and best-described history among all the Enhancers include ascorbic acid and muscle tissue, which may
trace elements; however, it was not until 1932 that the reduce Fe3þ to Fe2þ and bind it in soluble complexes, which
importance of Fe was finally confirmed with the evidence that are available for absorption [98].
inorganic Fe was needed for hemoglobin synthesis [79]. It is a

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key element in the metabolism of almost all living organisms, Fe and Reproduction
as it is involved in central cellular processes, including DNA
synthesis, respiration, and oxygen transport. The biological As previously mentioned, the effect on pregnancy of Fe
functioning of Fe depends upon its ability to readily accept or deficiency in the mother and, more specifically, its negative
donate electrons, interconverting between Fe3 þ and Fe2 þ impact on fetal growth and development, has been the focus of
forms, as it does in enzymes, such as cytochromes [80]. In many studies [100]. Pregnancy increases the Fe requirement to
the human body, Fe is an essential component of hundreds of nearly 6 mg/day by the second and third trimesters, due to the
proteins and enzymes [7], where it mainly exists in complex high growth rates of the placenta and the fetus, and the
forms bound to heme protein (hemoglobin or myoglobin), as expansion of the maternal red blood cell mass [101]. Few
heme compounds, as heme enzymes, or as nonheme com- studies have been published on the effect of Fe on female
pounds (flavin-Fe enzymes, transferrin, and ferritin) [81]. In fertility; however, the preliminary consensus is that a greater
hemoglobin, Fe is in the Fe2 þ form, whereas Fe transported on intake of Fe, either due to increased dietary intake or
transferrin in the blood, or stored intracellularly in ferritin, is in supplementation, increases fertility levels in women, with the
the Fe3þ form [80]. An understanding is emerging on how comment that the utilization by the ovary of this element is the

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these proteins, both individually and in unison, maintain likely reason for this observation [42]. In a large study
cellular and whole-body homeostasis of this crucial trace involving 18 555 women, Chavarro et al. [102] evaluated
element [82]. Fe hemostasis is a complex process, involving whether Fe supplement use or greater intake of total heme and
many proteins that respond not only to Fe load (intake and nonheme Fe is associated with lower risk of ovulatory
storage), but also to stimuli including inflammation, hypoxia, infertility. Women who consumed Fe in supplements had a
and anemia [83]. significantly lower risk of ovulatory infertility than women
Although low Fe intake and its poor bioavailability are who did not use Fe. More specifically, total nonheme Fe intake,
responsible for most anemia in industrialized countries, this consumed as multivitamins and Fe supplements, was inversely
accounts for only about half of the causes of anemia in associated with the risk of ovulatory infertility, whereas heme
developing countries [84]. In these regions, infectious and Fe intake was unrelated to ovulatory infertility in multivariable
inflammatory diseases, such as malaria, blood loss caused by adjusted analyses [102]. A recently published study to ascertain
parasitic infections, and nutrient deficiencies, including those the effects of severe Fe deficiency on fertility in female rats
for vitamins A, B2, B9, and B12, are also important causes [85]. found that there was a significantly lower conception rate and a
The role of Fe deficiency in detrimentally influencing disruption of estrus in an Fe-deficient group relative to the
pregnancy outcomes is well established, and governments controls [92]. These results suggest that the importance of Fe in
and researchers have focused a significant amount of attention female reproductive function begins well before pregnancy.
on this aspect [86–91]. There has been little research, however, There are few studies investigating the effect of Fe overload
into the relationship between Fe deficiency and unexplained
(toxicity) and fertility, and even less with regard to female
infertility [92].
fertility. However, there is a link between Fe overload and
female infertility [103, 104]. Excess Fe leads to reduced
Metabolism of Fe production of the hormones LH and FSH from the anterior
The fraction of Fe absorbed relative to the amount ingested pituitary, suggesting impaired oocyte maturation and low
is typically quoted in the range of 5%–35%, depending on the ovarian reserve [103]. In patients with beta thalassemia,
combination of foods ingested and the oxidation state of Fe multiple blood transfusions and increased gastrointestinal Fe
[81]. Fe absorption predominantly takes place in the duodenum absorption leads to Fe overload in the body [105] and infertility
and upper jejunum [93], and occurs by the enterocytes by [103], likewise in patients with hemochromatosis, a genetic
divalent metal transporter 1, a member of the solute carrier condition that leads to increased serum Fe levels and also sub-
group of membrane transport proteins. From here, it is fertility in women [104].
7 Article 86
 CEKO ET AL.

These selenoproteins play an important role in many biological

functions, such as antioxidant defense, formation of thyroid
hormones, DNA synthesis, fertility, and reproduction [112]. By
working synergistically with vitamin E, GPXs play an
important role in the body, catalyzing the conversion of
peroxides to nontoxic alcohols and thus protecting cells from
membrane damage and oxidative stress [113, 114]. The
concentrations of Se in whole blood and in plasma and/or
serum are related to dietary intake, with 50%– 60% of the total
plasma Se being present as selenoprotein P [108, 109], about
30% being present as GPX3, and the remainder incorporated
FIG. 4. Localization of Zn, Fe, and Se in a large bovine antral follicle. (a) into albumin as selenomethionine [115]. Low serum Se levels
H&E-stained serial section of a 15-mm-diameter healthy follicle. (b) The
corresponding RGB image was generated from XRF elemental distribution
have been linked to higher cancer risk based on evidence from

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maps and depicts the distribution of Zn (green), Fe (red), and Se (blue). *, observational studies conducted over a 40-yr period (see
vasculature; - - - - -, the separation between granulosa layer and the thecal reviews [116–119]. Thus, adequate Se intake may have an
interna; gc, granulosa cells. Scale bar ¼ 500 lm. Reprinted from Ceko et anticancer effect, due to its contribution to antioxidant function
al. Metallomics 2015; 7:71–82 [47] and reproduced by permission from or to immune system function, but the precise mechanism is
The Royal Society of Chemistry. uncertain. Weekley and Harris [117] highlight that epidemio-
logical studies have reported an inverse relationship between
Using S-XRF, we were able to determine the precise Se status and the incidence of various diseases, but subsequent
location and levels of Fe in bovine ovarian sections [35, 36]. studies of Se supplementation and disease prevention have
Bioaccumulation of Fe in the ovary is elevated in corpora lutea: presented mixed results, with concerns about this trace
low at early stages of development and high in mature corpora element’s toxicity [117].

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lutea. This would be consistent with Fe as a major component
of cytochrome P450 cholesterol side-chain cleavage, which is Bioavailability and Absorption of Se
elevated in mature bovine corpora lutea [106], and with the Fe The Se content of food is dependent upon the Se content of
in hemoglobin in the highly vascularized corpora lutea [76]. Fe
the soil in which the plants are grown or the animals are raised.
in capillaries of follicles, likely due to hemoglobin, can be seen
Se is found in highest amounts in organ meats, such as kidney
in Figure 4. Regressing corpora lutea and regressed follicles
and liver, while some seafood contains nearly as much [120].
(Fig. 3C) are also high in Fe, which probably represents
That said, plant foods, such as grains, legumes, and cruciferous
hemosiderin derived from the dead cells that contained the
vegetables, are a major dietary sources of Se in most countries,
steroidogenic cytochrome P450 enzymes. Fe levels are also
elevated in the walls of arterioles, and these levels are five where it enters the food chain predominantly as selenomethi-
times that of the levels within the lumen of the arterioles [36]. onine [109, 121]. Se, which is initially taken up from the soil
At least one other study has reported high Fe concentrations in and concentrated by plants, is absorbed in the small intestine
blood vessels and in smooth muscle cells imaged by XRF [77]. and incorporated into proteins by complex mechanisms that
They hypothesized that the elevated levels are owing to the remain to be fully elucidated [122]. Preintestinal absorption of

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presence of Fe in smooth muscle myoglobin. In addition to a Se is negligible, so the absorption operates mainly in the
clear higher density of Fe in the compartments discussed duodenum and caecum [112]. Similar to Fe, the mechanisms of
above, Fe is crucial for many basic processes in all ovarian intestinal absorption are different depending on the chemical
cells, including respiration, DNA synthesis, antioxidant form of the element. A recent study designed to investigate the
activities, and steroidogenesis, which is representative in the bioavailability of four Se species using an in vitro model of the
more dispersive distribution shown in Figure 4. intestinal barrier determined that the efficiency of Se
absorption decreased in the following order: selenomethionine,
SELENIUM methylselenocysteine, selenate (SeO42), and selenite (SeO32)
[123]. An earlier report presented by the U.S. Institute of
The trace element Se was discovered in 1817 by the Medicine, however, summarized that Se from the inorganic
Swedish physician and chemist Berzelius; however, its salts, SeO42 and SeO32, is more rapidly incorporated into
essentiality in mammals was not discovered until the 1950s GPX and other selenoproteins than Se from organic sources
[107]. Conclusive evidence for the essentiality of Se in humans containing selenomethioine. SeO32 is absorbed by simple
came with publication of the results of large-scale trials in diffusion, and its subsequent reduction is a well-characterized
China that showed the protective effect of Se supplementation metabolic pathway in animals [124]. The proportion of
on children and young adults suffering from Keshan disease, a ingested Se excreted is dependent upon dietary intake, with
cardiomyopathy endemic in regions with low soil Se levels high consumption resulting in high urinary and fecal excretion
[108]. Whole-body Se is about 15 mg, as estimated by direct and vice versa [125, 126]. In cases of dietary Se deprivation,
tissue analysis and radioisotope techniques, with the tissue the synthesis of some selenoproteins (e.g., GPX4) is prioritized
concentrations of this essential trace element being highest in over that of others [111]. Conversely, GPX1 is one of the most
the kidney and liver [109]. The most important biologically highly sensitive selenoproteins to changes in Se status, with
active compounds contain selenocysteine, where Se is levels of mRNA and protein dramatically reduced under low Se
substituted for sulfur in cysteine. The insertion of selenocys- conditions [127].
teine to form a selenoprotein is specified by the UGA codon in
mRNA under specific conditions [110, 111]. Se and Reproduction
In humans, the nutritional functions of Se are achieved by
its incorporation into 25 selenoproteins (including the gluta- Se has long been recognized in animal husbandry as being
thione peroxidases [GPXs], thioredoxin reductases and deio- essential for successful reproduction [2]. Of particular
dinases) that have selenocysteine at their active center [110]. importance to reproduction and pregnancy are the GPXs,
8 Article 86
 TRACE ELEMENTS IN OVARIES

which play a crucial role in reducing hydrogen peroxide confirmed a strong association between GPX1 and large,
(H2O2) and lipid peroxides to harmless products, thereby healthy follicles, suggesting a role for this antioxidant in
reducing cellular damage by reactive oxygen species (ROS) follicle dominance, protecting the dominant follicle from
[128]. The emerging role of Se in maintaining healthy increasing levels of ROS [47]. Furthermore, an additional
reproductive function has been extensively studied in males, study using human cumulus cells derived from cumulus-oocyte
but data pertaining to Se status and fertility in females are complexes collected for both in vitro fertilization and
sparse, with the majority of female-based studies having a intracytoplasmic sperm injection were assessed using RT-
tendency to focus on the role of this element in pregnancy PCR for GPX1 expression before fertilization [47]. These
[129–133]. cumulus cells retrieved from cumulus-oocyte complexes prior
Over 30 yr ago, Behne et al. conducted several studies in to embryo transfers that resulted in a pregnancy (n ¼ 12) had
male rats and observed that, during insufficient Se intake, the significantly higher expression of GPX1 compared with those
supply of this trace element to the testes was prioritized over that did not result in a pregnancy (n ¼ 18) [47]. Notwithstand-
other tissues. This suite of studies led the authors to postulate ing the fact that much further research is needed, these
the involvement of Se in the biosynthesis of testosterone [134,

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observations do suggest a strong role for selenoprotein GPX1
135], and it is now commonly known that testicular tissue in determining follicle growth, maturation, and dominance in
contains high concentrations of Se, predominantly as GPX4, both the cow and in women, and lead to further specific dietary
and that this element is essential for the formation and normal recommendations for Se and the role oxidative stress may play
development of spermatozoa [136, 137]. Some evidence in infertility.
suggests that increasing Se dietary intake increases antioxidant
GPX activity, thereby increasing male fertility [138]. In a study ZINC
of subfertile Norwegian men, the Se concentration of seminal
plasma correlated positively with the concentration of sperma- Zn is an essential element in the nutrition of human beings,
tozoa [139]. Scott et al. [140] supplemented subfertile men other animal species, and plants, and is required in every cell

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with 100 lg of Se per day for 3 mo and, in addition to their [7]. Zn plays an important role in growth and development, the
sperm motility significantly increasing, 11% of men receiving immune response, neurological function, and reproduction
the active supplement fathered a child compared with none in [146, 147]. Zn is needed for DNA and RNA synthesis and is
the placebo group. It should be noted, however, that a similar required at every step of the cell cycle, including proliferation,
study carried out over a similar length of time, but with double differentiation, and apoptosis [147]. Zn plays an important role
the daily dose of Se, showed no beneficial effect on sperm in the structure of proteins and cell membranes, and is an
motility in subfertile Polish men [141]. Closer observation of integral component of over 300 metalloenzymes, including
the form of Se administered in each of these studies indicates carbonic anhydrase, alcohol dehydrogenase, thymidine kinase
that they differed, with the first being selenomethionine versus carboxypeptidase, glutamate dehydrogenase, lactate dehydro-
SeO32 in the latter Polish study. This highlights that it is not genase, and alkaline phosphatase [5, 148]. Zn additionally
necessarily the quantity of the trace element ingested that participates in the synthesis and degradation of carbohydrates,
matters with regard to exerting a beneficial biological effect, lipids, proteins, and nucleic acids [3]. Furthermore, Zn finger
but more so its bioavailability and subsequent absorption by proteins, formed when Zn atoms become tetrahedrally
the body. coordinated with histidine and cysteine [149], regulate the

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With regard to Se status and fertility in females, Paszkowski expression of many genes [150, 151], and Zn has been shown
et al. [142] completed a study of 135 follicular fluid samples to have antiapoptotic and antioxidant properties [152–154].
collected from patients during transvaginal oocyte retrieval, Inadequate intake of this trace element in humans and other
and found that patients with unexplained infertility had animal species results in immunodeficiency, increased numbers
significantly decreased follicular Se concentrations compared of infections, increased severity of infections, stunted growth,
with those with other known causes of infertility [142]. A case- and delayed sexual maturation, as summarized by Qureshi et al.
controlled study from Turkey found lower Se concentrations in [7].
the serum and follicular fluid of women undergoing in vitro
fertilization treatment compared with age-matched, nonpreg- Bioavailability and Absorption of Zinc
nant control women [143], and a recent study, also from
Turkey, found that plasma Se levels were significantly lower in Zn is found as organic complexes with protein in meats and
women with polycystic ovary syndrome relative to the control as inorganic salts in plant foods [155, 156]; however, no
group, indicating that this element may play a role in the absorption studies have been conducted in humans to
pathogenesis of polycystic ovary syndrome related to hyper- determine whether these forms of Zn differ in their bioavail-
androgenism [144]. Finally, women presenting with unex- ability [157]. Based on limited published data on the absorption
plained infertility or premature ovarian failure were found to of Zn from supplements ingested by humans, it appears that Zn
have significantly increased serum levels of the ovarian gluconate, Zn citrate, and Zn sulfate are absorbed to a similar
autoantibody protein, Se-binding protein-1 [145]. Ultimately, extent, and that Zn oxide is slightly less well absorbed [158,
Se plays a significant role in living organisms, and owing to its 159]. A recent study in rats, however, found all four of these
contribution to the formation of selenoproteins combined with species to be equally bioavailable [160]. Similar to the
its antioxidant action, a low Se status in the body induces a low aforementioned trace elements (Cu, Fe, and Se), inadequate
resistance to free radical damage [112]. dietary intake of Zn can arise from low intake, poor
Our investigations into the bioaccumulation of Se in ovarian bioavailability, or a combination of these dietary factors.
tissue using S-XRF demonstrated that Se was consistently Low dietary Zn intake is often associated with developing
localized to the granulosa cell layer of large, healthy follicles countries, owing to the prohibitive cost of Zn-rich foods (i.e.,
(Fig. 4) [36]. In addition, the increase in Se in these follicles meat, poultry, and fish) or religious restrictions [161]. The total
was likely due to the increased expression of selenoprotein Zn content of one’s diet is influenced, not only by the range of
GPX1, which was significantly upregulated compared to small food items selected and their relative bioavailability, but also
healthy or atretic follicles. Western blot analyses also by the degree of refinement of any constituent cereals. In
9 Article 86
 CEKO ET AL.

addition, a diet rich in saturated or unsaturated fats tends to Kim et al. investigated the abundance of Zn throughout the
dilute the uptake of Zn [3]. ovarian cycle in mice using S-XRF [189]. Interestingly, during
Zn is absorbed in the small intestine by a carrier-mediated the period of meiotic maturation, between the LH surge and
mechanism [162], with ongoing research over the last decade ovulation, there was a 50% increase in total Zn within the
indicating that its transport across membranes is mediated by ovary [189]. Moreover, within the unfertilized egg, there is a
two subfamilies of mammalian Zn transporters, ZnT (Slc30) polar distribution of Zn, which the authors suggest are Zn-
and Zip (Slc39) [163–166]. Following absorption through the enriched cortical granules that may play a role in the hardening
enterocyte, dietary Zn is transported into the circulation and of the zona pellucida required to block polyspermy at
then to the tissues, where it is needed. Active transport of Zn fertilization [189]. In a more recent study, Tian et al. [190]
into portal blood is mediated by metallothionein [167]. Zn is demonstrated that Zn deficiency in mice before fertilization led
released from food as free ions during digestion and the to reductions in placental and fetal development, highlighting
liberated ions may then bind to endogenously secreted ligands the role of trace elements and the dependency that the embryo
before their transport into the enterocytes in the duodenum and has on the integrity of the oocyte predecessor. Taken together,
these studies demonstrate that Zn has a vital role in ovarian

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jejunum [16, 168]. In contrast to Fe, the body regulates Zn
homeostasis through gastrointestinal secretion and excretion of function, and cytoplasmic levels within the oocyte are crucial
endogenous Zn, in addition to the absorption of exogenous Zn for early embryo and placental development [189, 190].
[169, 170]. Loss of Zn through the gastrointestinal tract Similar to the alleged role of Se in reproduction, Zn is
accounts for approximately half of all Zn eliminated from the important for several antioxidant functions that provide
body, with pancreatic and biliary pathways being other modes protection of cells against oxidative and electrophilic stress
of excretion. This is an important process in the regulation of caused by ROS. Many investigations have shown evidence for
Zn balance [171], and similar to Cu and Se, with higher Zn the role of ROS in the physiology and pathology of both male
intake balanced by more secretion and vice versa [172]. and female reproductive functions [191, 192], suggesting that
As alluded to earlier, plant-based foods are the major source the focus should shift to elucidating the biochemical roles these

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of dietary Zn in many low-income countries. Of the plant- trace metals play in the process.
based foods, cereals and legumes contain high levels of phytic Our investigations using S-XRF into the accumulation of Zn
acid and their magnesium, calcium, and potassium-associated in bovine ovarian tissue indicate that the highest Zn levels
salts, called phytates. While amino acids and peptides in chyme always corresponded to either the walls of arterioles or to
facilitate the absorption of Zn, the binding of Zn by phytate and capillaries (Figs. 3 and 4), and was associated with above-
dietary fibers forms insoluble complexes, rendering the Zn background Fe concentrations [36]. Although at lower levels
relatively unavailable [173, 174]. than blood vessels, Zn was also elevated in the thecal and
granulosa cell layers relative to the stromal tissue. Healthy
Zinc and Reproduction follicles contain an average of three times more Zn than the
regressed follicles. Using principal component analyses, a
Countless studies have focused on the role of Zn in male relationship was found to exist between levels of Br and Zn,
fertility, paying particular attention to its role in male sex showing that the healthy and early atretic follicles are different
hormone synthesis, as well as the resultant impact on sperm from atretic and regressed follicles, suggesting that the roles of
production and motility. Zn content is high in the adult testis, these elements in healthy follicles may differ between growth

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and the prostate has a higher concentration of Zn than any other and atresia [35].
organ of the body [1]. Zn deficiency first impairs angiotensin-
converting enzyme activity, which leads to depletion of FUTURE DIRECTIONS
testosterone and inhibition of spermatogenesis. Defects in
spermatozoa are frequently observed in Zn-deficient rats [175, The recent new capabilities to both localize and quantitate
176]. In females, Zn also seems to be important in trace elements in tissues will allow us to identify where and
reproduction, but relatively few investigations have been when trace elements accumulate in reproductive tissues during
performed [45]. Shaw et al. [177] found that Zn deficiency in their development and in disease states. Just as the recent
female rabbits resulted in sexual disinterest by their male studies in the ovary have demonstrated, this may lead to
counterparts. Another study suggested that Zn deficiency in discoveries of new roles played by these elements in
female rats led to abnormal estrous cycles [178], and the effects reproduction.
of Zn deficiency in two species of monkeys led the authors to
conclude that normal reproduction was impaired in both the ACKNOWLEDGMENT
species through abnormal ovarian development [179]. Over 30 We thank the Australian Synchrotron, Victoria, Australia for their
yr ago, Sato et al. [180] reported that even marginal Zn support and for the use of their facilities.
deficiency could affect oocyte maturation by doubling the
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