Document Type : Original Article
Previous studies have shown that lipopolysaccharide
(LPS) affects brain development and results
in behavioral disorders in many species. Epidemiological
researches have reported that maternal
bacterial and viral infections during pregnancy
represent a risk factor for several neuropsychiatric
disorders with a presumed neurodevelopmental origin.
Studies using animal models have also shown
that both bacterial and viral infections in utero can
cause a spectrum of neuropathological and behavioral
abnormalities in offspring (
Previous studies have shown that exposing
pregnant female mice to stressors during the last
week of pregnancy, reprograms the hypothalamicpituitary-
adrenal (HPA) axis and enhances behavioral
responses to psycho stimulants (
Repeated exposure of pregnant mice to stressful
environments during pregnancy has been found to
induce deficits in cognitional behaviors (
Since previous studies considered, bacterial infections during pregnancy as a risk factor for brain and behavioral development in the fetus, this current study aims to investigate the effects of prenatal exposure to bacterial LPS on the development of reproduction-related behaviors and serum concentration of luteinizing hormone (LH), Follicle-stimulating hormone (FSH) nd testosterone in adult male offspring.
In this experimental study, female and male NMRI mice obtained from Pasteur Institute of Iran, were aged between 10-12 weeks at the time of testing. Animals were housed in groups of 4 per cage in a room with a 12:12 hour light/dark cycle (lights on 07:00 am) under a controlled temperature (23 ± 1˚C). Animals had access to food and water. Breeding began after 2 weeks of acclimatization to the new animal holding room. The breeding procedure and the verification of pregnancy have been fully described in previous studies (
Pups were weaned on postnatal day 21 (PD 21), and offspring housed (four animals from the same treatment/cage) and maintained on a standard animal house condition. Adult male offspring randomly chose for each test were distributed into control and experimental groups (n=10/group, two pup each litter for the adulthood behaviors) before starting each test (
All animal experiments have been carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH).
In order to model a physiological maternal infection, at the 10th gestational day, pregnant mice were administered with intra-peritoneal injection of low doses of LPS (from Brucella abortus, Pasteur Ins., Tehran) which have been shown to produce optimal fever and cytokine induction in the mice, while having limited impact on maternal and pup survival (i.e. 1, 5 and 10 μg/kg of LPS, N=7/group) (
Maternal serum was prepared 2 hours after injection of saline or LPS by centrifugation at 15000 g for 5 min, aliquoted and then stored at -80˚C until the cytokine assays were performed. Concentrations of), interleukin-1beta (IL-1β) (Immuno-Biological Laboratories, IB49700, USA), interleukin-6 (IL-6) (BioSource International, CA 93012, USA), and Tumor necrosis factor-alpha (TNF-α) (Ucytech, CT 302, Netherlands) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturer’s instructions. All samples and standards were assayed in duplicate.
Testosterone, LH and FSH hormones of adult male offspring were assayed by solid phase ELISA kits (Demeditec Diagnostics Ltd., Germany), based on the principle of competitive binding and according to the manufacturer’s instruction.
The microtiter wells are coated with an antibody directed towards a unique antigenic site on the hormone molecule. Endogenous hormone of a serum sample competes with a hormone horseradish peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate is washed off. The amount of bound peroxidase conjugate is proportionally reverse the concentration of hormone in the sample. After addition of the substrate solution, the intensity of color developed is proportionally reverse the concentration of hormone in the serum sample.
Adult male offspring were randomly chosen for behavioral testing. Receptive female mice were used to test male reproductive activity (Sniffing, Following, Mounting, Coupling) in such a way that males were placed in the female’s acrylic cage (25 cm×25 cm×40 cm; L×W×H) containing wood chips with food and water provided.
Before studying sexual behavior, control (prenatally exposed to saline) and LPS treated (prenatally exposed to LPS) male offspring were separately placed in a cage with a sexually experienced male and a receptive female to have prior learning or experience. Early morning of assessing day, sexually naive males were separated and maintained separately until that evening. Every naive male, currently sexually experienced, was given 60 minutes to accompany a receptive female, during which male behaviors were assessed and compared (
Since data displayed normality of distribution and homogeneity of variance, one-way ANOVA and Tukey Post Hoc test (SPSS 16) were used for comparison between the effects of different doses of extract with control.
As shown in
Effects of intra peritoneal injection of saline (0.05 ml/mice) or LPS (1, 5 or 10μg/Kg) on serum level of IL-1β (A), IL-6 (B) and TNF-α (C) in pregnant dams. Each bar is mean ± SE.
*p<0.05, **p < 0.01 and ***p < 0.01, when compared to the saline treated group (N=7).
Figure 2 shows the effects of prenatal LPS administration on serum levels of IL-1β, IL-6 and TNF-α in male offspring. As shown in the
The results of this research suggest that prenatal LPS exposure decreases sexual behavior components including coupling (p<0.01), following (p<0.01), mounting (p<0.01), and sniffing (p<0.001), significantly in comparison with the control group (
Measuring pituitary-gonadal hormones after assessing behavioral components in prenatally LPS exposed mice showed that testosterone (p<0.001) and LH (p<0.01) concentrations of serum were significantly reduced (
Effects of prenatal exposure of saline (0.05 ml/mice) or LPS (1, 5 or 10μg/Kg) on serum level of IL-1β (A), IL-6 (B) and TNF-α (C) in the male adult offspring. Each bar is mean ± SE (N=7).
Effects of prenatal LPS exposure on behavioral components of adult Male offspring, in comparison with the control group (mean ± SE)
|Control||LPS 1(µg/rat)||LPS 5(µg/rat)||LPS 10(µg/rat)|
|16 ± 1.6||8.18± 0.78*||7.38± 0.86**||6.10± 1.1***|
|10.78 ± 0.72||6.87± 1.5||4.81± 8.8**||4.25± 0.64**|
|4.31 ± 0.6||1.7 ± 0.67||1.25± 0.44*||1.25± 0.31*|
|0.8 ± 0.1||0.5 ± 1.16||0.2 ± 0.13**||0.3 ± 0.21**|
*p<0.05, **p<0.01 and ***p<0.001 when compared to the saline treated group.
Effects prenatal exposure to saline (0.05 ml/mice) or LPS (1, 5 or 10ìg/Kg) on serum level of Testosterone (A), LH (B) and FSH (C) in adult male offspring. Each bar is mean ± SE. *p<0.05, **p<0.01 and ***p<0.01, when compared to the saline treated group (N=10).
It is clear that maternal bacterial infections and immune challenges during pregnancy have distinct effects on the development of central nervous and endocrine systems in the offspring, that may affect behavior (
Results of the present study show that prenatal exposure of adult male NMRI mice with low doses of Brucella abortus LPS inhibits sexual behaviors and decreases serum level of testosterone and LH hormones. Our results also demonstrate that LPS exposure during gestation increased serum concentration of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. However, prenatal LPS treatment has no significant effect on serum concentrations of IL-1, IL-6 and TNF-α in the male adult offspring.
It is well known that lipopolysaccharide as a bacterial outer membrane component, induces production of several pro-inflammatory cytokines (
A previous study carried out by Bernardi et al. (
Increased GABAergic inhibitory activity may also contribute to the effects of prenatal stress on behavioral alteration in adulthood.
Previous studies have shown that prenatal exposure to stress and elevated levels of corticostrone affect the GABAergic system of a developing brain. Prenatal exposure to stressful environment and elevated activity of HPA-axis alters expression of GABA-A receptor subunit mRNA levels in the brain.
Prenatal exposure of the fetus to high levels of corticostrone affects mRNAs expression for glutamic acid decarboxylase (GAD) isoforms, the enzyme that converts glutamate to GABA. Stone and co-workers demonstrate that prenatal increase of corticostrone level increase GAD67 mRNA in the brain’s hippocampus. Therefore, prenatal activation of HPA axis by LPS may increase the activity of GABAergic system in the brain regions such as hippocampus that are involved in the modulation of behavior (
Our results indicate that LPS administration on the 10th gestational day influences sexual behavior and expression of the pituitary-gonadal hormones of male offspring. Therefore, this study has identified that bacterial lipopolysaccharide exposure during pregnancy and the ensuing cytokine changes, can affect development of neural systems involved in reproduction of animals.