Reproductive biology study of dynamics of female sexual hormones: A 12-month exposure to lead acetate rat model

Abstract

In human and animal organisms, lead can cause reproductive problems beginning with pregnant females. The reproductive axis is particularly sensitive to lead, its influence resulting in a delayed sexual maturity due to biosynthesis suppression of the sexual steroids. An animal model study was carried out on 28 white Wistar adult female rats, divided into 3 experimental (E) groups that were exposed for 12 months to lead acetate in drinking water as follows: 50 ppb Pb (E1), 100 ppb Pb (E2), and 150 ppb Pb (E3), with a control group (M) that received unleaded tap water. Levels of FSH, LH, estradiol, progesterone, and testosterone were evaluated in the proestrus phase by ELISA technique. Data obtained were compared by one-way ANOVA with Bonferroni correction. As a conclusion, compared to the M group, we can ascertain that lead acetate administered over a long-term period to female rats determines (with the exception of estradiol and progesterone), in direct correlation with the exposure levels, the following: significantly decreased FSH, but still within physiological limits of serum levels; significantly higher serum levels of LH; significantly decreased serum levels of estradiol and progesterone; and significantly higher serum levels of testosterone

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581
http://journals.tubitak.gov.tr/biology/
Turkish Journal of Biology
Turk J Biol
(2014) 38: 581-585
© TÜBİTAK
doi:10.3906/biy-1402-50
Reproductive biology study of dynamics of female sexual hormones: a 12-month
exposure to lead acetate rat model
Eugenia DUMITRESCU1, Romeo Teodor CRISTINA1,*, Florin MUSELIN2
1Department of Veterinary Pharmacology and Pharmacy, Faculty of Veterinary Medicine Timișoara,
Banat University of Agronomical Sciences and Veterinary Medicine, Timișoara, Romania
2Department of Toxicology, Faculty of Veterinary Medicine Timișoara,
Banat University of Agronomical Sciences and Veterinary Medicine, Timișoara, Romania
* Correspondence: rtcristina@yahoo.com
1. Introduction
e scientic concerns of the last decade emphasize the
importance and timeliness of reproductive health research
in animals and humans. Among the numerous causes of
reproductive disorders, authors consider, as an important
threat, the more and more frequent presence of potential
toxic risk disruptors (e.g., industrial contaminants, heavy
metals, pesticides, organic solvents, phthalates) in dierent
reproductive pathologies. Among these, lead can act as an
important disruptor (EPA, 1997; Altundoğan et al., 1998).
Lead can be found in the soil crust, in mineral form
as galena, anglesite, cerussite, mimetite, pyromorphite,
linarite, vanadinite, and wulfenite (Humphreys, 1988).
e reproductive axis is particularly sensitive to lead,
its inuence resulting in a delayed sexual maturity due to
biosynthesis suppression of the sexual steroids (Ronis et
al., 1998). In this respect, Nicolopoulou-Stamati and Pitsos
(2001) conrmed that lead can certainly inuence the
female endocrine balance, the estrous cycle and fertility
being very sensitive to this reproductive disruptor. Unlike
other metals, lead has no physiological role in the body
and there is no known accepted minimum level that
could
be considered as nontoxic for humans and animals. In
organisms lead can cause reproductive problems beginning
with pregnant females (such as premature birth, abortion,
or fetal resorption), but debilitated and young individuals
are the most aected, lead inuencing their viability,
normal growth, and development (Wide, 1985; Téllez-Rojo
et al., 2004). Peripubertal exposure in females or for long
periods determines delay in vulval opening and puberty
installation, all associated with low insulin-like growth
factor 1 (IGF-1), luteotropic hormone, and estradiol serum
levels (Pinon-Lataillade et al., 1995; Dearth et al., 2004).
Nampoothiri and Gupta (2006) demonstrated that
lead is also involved in the gonadotrophic metabolism,
disrupting the activity of steroidogenic enzymes from
ovarian cell granulose. As a result, lead will aect the
cellular membrane through free radicals, inducing lipid
peroxidation.
Silberstein et al. (2006) showed that accumulation
of lead in small amounts over long periods in the ovary
will cause irreversible folliculogenesis, with the presence
of atretic follicles and heavy diminution of the primary
follicles.
Abstract: In human and animal organisms, lead can cause reproductive problems beginning with pregnant females. e reproductive
axis is particularly sensitive to lead, its inuence resulting in a delayed sexual maturity due to biosynthesis suppression of the sexual
steroids. An animal model study was carried out on 28 white Wistar adult female rats, divided into 3 experimental (E) groups that
were exposed for 12 months to lead acetate in drinking water as follows: 50 ppb Pb (E1), 100 ppb Pb (E2), and 150 ppb Pb (E3), with a
control group (M) that received unleaded tap water. Levels of FSH, LH, estradiol, progesterone, and testosterone were evaluated in the
proestrus phase by ELISA technique. Data obtained were compared by one-way ANOVA with Bonferroni correction. As a conclusion,
compared to the M group, we can ascertain that lead acetate administered over a long-term period to female rats determines (with the
exception of estradiol and progesterone), in direct correlation with the exposure levels, the following: signicantly decreased FSH, but
still within physiological limits of serum levels; signicantly higher serum levels of LH; signicantly decreased serum levels of estradiol
and progesterone; and signicantly higher serum levels of testosterone.
Key words: Reproductive biology, females, hormones, rats, lead acetate
Received: 21.02.2014 Accepted: 08.05.2014 Published Online: 05.09.2014 Printed: 30.09.2014
Research Article

DUMITRESCU et al. / Turk J Biol
582
In Romania, where pollution from the lead industry
still exists, information regarding the impact of lead on
reproductive function is still needed and the information
provided here will be useful for the reproductive biology
eld, justifying the present study.
2. Materials and methods
e present research was performed in compliance with
good laboratory practice; in accordance with the European
Convention principles for the protection of vertebrate
animals used in experimental and other scientic
purposes, adopted in 1986 in Strasbourg (Council of
Europe, 1986); in accordance with the 2010/63/EU
directive of the European Parliament and of the European
Council adopted 22 September 2010 on the protection of
animals used for scientic purposes (European Council,
2010); in accordance with Romanian law for animal
experimentation (Romanian Government, 2002); and
with the approval of the Scientic Ethics Committee of the
Faculty of Veterinary Medicine Timisoara.
2.1. Animals
e study was carried out on 28 white Wistar preadult
female rats (at 35 days and 220 g average weight). Animals
were purchased from the authorized biobase of “Victor
Babeș” University of Medicine and Pharmacy, Timișoara,
Romania. e animals were acclimatized for 7 days,
maintained in standard cages with controlled temperature
and humidity. For this purpose, animals were housed in
polycarbonate cages with 750 × 720 × 360 mm (L × W ×
H) dimensions and wood shavings were used as bedding.
e environmental temperature was maintained at 20
± 2 °C and relative humidity was 55 ± 10%. During the
acclimatization period, the light cycle was 12 h light and 12
h dark. Nonsterile pelleted diet (Biovetimix, code 140-501,
Romania) and water were oered ad libitum.
Rats were divided into 3 experimental groups as
follows: E1 (50 ppb Pb), E2 (100 ppb Pb), and E3 (150
ppb Pb), exposed continuously for a 12-month period to
lead acetate in drinking water, with a control group (M)
that received unleaded tap water. At 24 h aer the last
administration, the rats were euthanized and examined
according to the standard procedure during necropsy
(OECD, 2011). Euthanasia was performed by overdose of
anesthetic agents using the following association: ketamine
(300 mg kg bw–1) + xylazine (30 mg kg bw–1) (Pierce, 2006).
2.2. Methodology
Levels of follicle-stimulating hormone (FSH), luteinizing
hormone (LH), estradiol, progesterone, and testosterone
were evaluated in the proestrus phase. e sexual
hormones were determined by ELISA technique at Tody’s
Laboratories, Bucharest (ISO 170025 accredited).
2.3. Statistical analysis
Obtained data were analyzed using GraphPad Prism 5.0
(GraphPad Soware, USA). e data in dierent groups
were compared by one-way ANOVA with Bonferroni
correction. Dierences were considered to be signicant at
P < 0.05, P < 0.01, and P < 0.001.
3. Results
e values of serum hormones aer 12 months of exposure
are summarized in Figures 1a–1e. In the control group (M)
and in the exposed (E) groups, the FSH serum level was
within physiological limits (up to 500 ng/mL), toward the
inferior limit.
3.1. FSH levels
Exposure to lead caused signicant (P < 0.01) decrease of
FSH level compared to the M group and directly (P < 0.01)
correlated with the exposure level: E1 vs. M: –55.64%; E2 vs.
M: –67.72%; E3 vs. M: –88.05%; E2 vs. E1: –27.25%; E3 vs. E2:
–62.99%; E3 vs. E1: –73.08%.
3.2. LH levels
LH level was within physiological limits (35 ng/mL) in the
control group, while in experimental groups the LH level
was signicantly (P < 0.01) higher compared to the M group
and directly (P < 0.01) correlated with the exposure level:
E1 vs. M: +40.31%; E2 vs. M: + 66.77%; E3 vs. M: +169.24%;
E2 vs. E1: +18.86%; E3 vs. E2: +61.43%; E3 vs. E1: +91.89%.
3.3. Estradiol serum levels
e serum level of estradiol was at the inferior limit of the
physiological values (up to 50 ng/mL) both in M and the
E groups. Exposure to lead caused signicant (P < 0.01)
decrease of estradiol serum level in comparison to the
M group (E1 vs. C: –32.08%; E2 vs. C: –67.93%; E3 vs. C:
–88.77%) and inversely signicantly (P < 0.01) correlated
with exposure level (E2 vs. E1: –37.89%; E3 vs. E2: –73.20%;
E3 vs. E1: –83.43%).
3.4. Progesterone serum levels
e level of progesterone was within physiological limits
(up to 60 ng/mL) in M and the E groups. Exposure to lead
caused signicant (P < 0.01) decrease of serum progesterone
level compared to the M group: E1 vs. M: –12.08%; E2 vs. M:
–33.36%; E3 vs. M: –44.20%. Progesterone in the E groups
was inversely and signicantly (P < 0.01) correlated with
exposure level: E2 vs. E1: –24.20%; E3 vs. E2: –16.26%; E3 vs.
E1: –36.52%.
3.5. Testosterone serum levels
No references regarding physiological serum testosterone
limits for female rats were found. In our case, serum
testosterone level was signicantly (P < 0.01) higher in the E
groups compared to the M group (E1 vs. M: +72.72%; E2 vs.
M: +163.63%; E3 vs. M: +200.00%) and in direct correlation
(P < 0.01) with the exposure level (E2 vs. E1: +52.63%; E3 vs.
E2:+13.79%; E3 vs. E1: +73.86%).

DUMITRESCU et al. / Turk J Biol
583
4. Discussion
Gonadal activity is under the control of both the
hypothalamus and the anterior pituitary gland, the latter
being responsible for producing the hormones that are
very important for the gonads’ control (Cunningham
and Klein, 2007). FSH and LH are synergistic hormones
in folliculogenesis and ovulation in the ovary. ey play
a critical role in maintaining the ovarian cycle, governing
follicle recruitment and maturation, steroid genesis,
completion of ova maturation, ovulation, and luteinization.
e low levels of FSH can be explained by the fact that it is
very dicult to observe the FSH secretion
peak due to its
Figure 1. Dynamics of serum FSH (a), LH (b), estradiol (c), progesterone (d), and testosterone (e) levels in the studied group.
M
E 1
E2
E 3
0
50
100
150
200
250
a). Dynamics of FSH levels
M
E 1
E2
E 3
0
20
40
60
80
100
ng/mLng/mL
ng/mLng/mL
ng/mL
b). Dynamics of LH levels
M
E1
E 2
E3
0
2
4
6
c). Dynamics of estradiol levels
M
E1
E2
E 3
0
10
20
30
40
50
d). Dynamics of progesterone levels
M
E1
E 2
E3
0.0
0.1
0.2
0.3
0.4
e). Dynamics of testosterone levels

DUMITRESCU et al. / Turk J Biol
584
very short discharge period and because in the proestrus
phase the FSH levels are extremely low (Maeda et al., 2000).
Bibliographical information regarding FSH dynamics
under lead impact are scarce, though Foster et al. (1996)
ascertained that FSH levels signicantly decreased aer
a 10-year lead exposure in monkeys, which conrms
the inhibition of ovarian function due to lead. Eects on
circulating sex steroids were accompanied by variable eects
on levels of circulating LH, pituitary LH, and pituitary LH
‘beta’-mRNA, suggesting a dual site of lead action: one at
the level of the hypothalamic pituitary unit, and another
directly at the level of gonadal steroid biosynthesis (Ronis
et al., 1996).
We observed that the LH level increased signicantly in
comparison to the control group (M) and it was in direct
correlation with the exposure level. e increase of LH
level over the physiological limits could be explained by
the low level of estradiol and progesterone observed by us.
Doumouchtsis et al. (2009) stated that in the subjects
exposed in the short term to lead, high levels of FSH and
LH are associated with normal testosterone concentrations.
e authors also argued that lead will accumulate in the
ovarian granulose cells, causing delays in growth and
development and infertility in women.
Qureshi and Sharma (2012) showed that lead salts can
inhibit the FSH release, leading to atrophy and reduced
ovarian secretion of progesterone. Additionally, Dearth et
al. (2002) claimed that exposure to lead will result in delayed
sexual maturity installation associated with suppression of
serum levels of IGF-1, LH, and estradiol.
Estradiol is considered to be the steroid hormone with
the biggest inhibitory capacity over LH secretion in rats
(Freeman, 1994). Moreover, Maeda et al. (2000) reported the
existence of a negative feed-back produced by the estrogens
and progesterone secreted by ovaries over LH secretion
from late estrus up to the early proestrus phase. In our case,
the estradiol levels were at the inferior physiological limit,
even in the case of the control group (M), contradicting
earlier observations that in proestrus the estradiol level is
high. e possible explanation of what we have found could
be that the LH high levels beyond the physiological limits
as ascertained by us in the experimental lots are linked (P
< 0.01) with the inhibitory eect exerted by this hormone
upon hormonal balance. In our case, we found very low
estradiol levels being proportionally inverted (P < 0.01)
with those of LH, this nding having been also presented
by other authors (Freeman, 1994; Taupeau et al., 2001).
Our results obtained on hormonal dynamics are
dierent from those presented by Wiebe et al. (1998), who
armed that exposure of pregnant females to lead does not
have a signicant inuence on estradiol level.
Progesterone plays an important role inuencing the
length of sexual cycle in rodents, with progesterone and
estrogens working in a synergistic way (Westwood, 2008).
e progesterone levels in our case were signicantly
lower in the experimental groups than in the control group
(M). is is in agreement with Freeman’s (1994) nding
that, in the same period of physiological conditions, the
progesterone peak in proestrus is determined by the LH
secretion: the progesterone level will decrease and the LH
level will increase, probably as the result of lack of or delayed
proper/optimal progesterone secretion response by the
ovarian preovulatory follicles and as a nal consequence
of lead’s eect on ovary histoarchitectonics. Some authors
argued that lead exposure determines decreased serum
progesterone (Foster, 1992).
When it comes to the testosterone dynamic during
sexual cycles in female rats, no reference values were
found by us for this species. Furthermore, no information
was found in the bibliographical sources regarding lead’s
inuence on testosterone in female rats. In our case, we
have found that the tendency of testosterone dynamics was
that of a signicant increase (P < 0.01) in comparison to
the control group (M) and in direct association with the
exposure level. In Ryan’s (1982) opinion, the increase of
testosterone levels in women can be explained by lead’s
inhibitor activity on aromatase cytochrome P-450, an
enzyme necessary to bioconvert androgens into estradiol.
As a conclusion, we can state that lead acetate
administered over a long-term period to female rats
determines the following, directly and signicantly
correlated (with the exception of estradiol and
progesterone, inversely correlated) with the exposure level:
signicantly decreased of FSH serum levels, but within
physiological limits, as compared to the control group M;
signicantly higher LH serum levels as compared to the
M group; signicantly decreased estradiol serum levels
as compared to the M group; signicantly decreased
progesterone serum levels as compared to the M group;
and signicantly higher testosterone serum levels as
compared to the M group.
Extensive studies about the correlation between
environmental quality and health and between life quality
and health have become a priority for many research
teams, such as Karakaş et al. (2013) and Polat et al. (2013),
who successfully used rodent models to demonstrate their
hypotheses. Here we have conrmed the advantage of the
rat model and have shown that this species is well suited
for such research on hormonal and reproductive disorders.
Acknowledgment
is work was carried out as part of the project “Postdoctoral
School of Agriculture and Veterinary Medicine”,
POSDRU/89/1.5/S/62371, co-nanced by the European
Social Fund through the Sectoral Operational Programme
for Human Resources Development 2007–2013.

DUMITRESCU et al. / Turk J Biol
585
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