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Edem Ekpenyong Edem*, Blessing Uyo Nathaniel, Kate Eberechukwu Nebo,
Abiola Oluwatosin Obisesan, Ayodeji Augustine Olabiyi, Elizabeth Toyin Akinluyi
and Azeez Olakunle Ishola
Lactobacillus plantarum mitigates sexual-
reproductive deficits by modulating insulin
receptor expression in the hypothalamic-
pituitary-testicular axis of hyperinsulinemic mice
https://doi.org/10.1515/dmpt-2021-1000195
Received January 1, 2021; accepted April 5, 2021;
published online May 17, 2021
Abstract
Objectives: Hyperinsulinemia increases the risk factor
of diabetes and infertility at a manifold. Lactobacillus
plantarum has several medical significances with limited
reports. Hence, this study assessed the effect of L. plantarum
on sexual-reproductive functions and distribution of insulin
receptors in the hypothalamic-pituitary-testicular axis of
hyperinsulinemic mice.
Methods: Forty male adult mice were divided into five
groups as follows: control, high-fat diet (HFD) +streptozo-
tocin (STZ), therapeutic, co-administration group type 1
(CO-AD) and probiotics. They were either simultaneously
exposed to an HFD and L. plantarum treatment for 28 days
with a dose of STZ injection to induce hyperinsulinemia on
day 28 or treated with L. plantarum for 14 days, and
following induction of hyperinsulinemia. Mice were sub-
jected to a sexual behavioural test and thereafter sacrificed
under euthanasia condition. Blood, brain and testes were
collected for biochemical and immunohistochemical
assays.
Results: Treatment with L. plantarum ameliorated repro-
ductive hormones activity disruption, sexual behavioural
defects, antioxidant imbalance, insulin dysregulation
and lipid metabolism dysfunction following exposure
to HFD +STZ when compared to the hyperinsulinemic
untreated mice.
Conclusions: Taken together, data from this study reveal
that L. plantarum abrogated hyperinsulinemia-induced
male sexual and reproductive deficits by modulating
antioxidant status, lipid metabolism and insulin signalling
in the hypothalamic-pituitary-testicular axis of mice.
Keywords: hyperinsulinemia; hypothalamic-pituitary-
testicular axis; insulin-like growth factor-1 receptor;
Lactobacillus plantarum; seminal parameters; sexual
behaviour.
Introduction
Hyperinsulinemia, often associated with type 2 diabetes,
although not classically referred to as diabetes is a result of
excessive amounts of insulin in the blood when compared
to the insulin level of non-diabetic individuals. This results
in insulin resistance, the primary cause of hyper-
insulinemia, with the pancreas compensating by produc-
ing more insulin. If not treated properly, this type of insulin
resistance can progress to type 2 diabetes [1], a condition
whereby the pancreas is unable to secrete sufficient
amounts of insulin required for maintenance of physio-
logic blood glucose levels. Insulin is a major chemical
messenger that is critically needed for cellular glucose
metabolism [2]. Pancreas, being the main source of insulin
available for cellular homeostasis is composed of clusters
of cells called islands or islets (of Langerhans). These
endocrine cells produce the hormone insulin and deter-
mine the amount needed based on the body’s blood
glucose levels. The higher the level of glucose, the more
insulin goes into production to balance sugar levels in the
*Corresponding author: Edem Ekpenyong Edem, Department of
Anatomy, College of Medicine and Health Sciences, Afe Babalola
University, P.M.B. 5454, Ado-Ekiti, Ekiti State, Nigeria, Phone:
+2347037402143, E-mail: ee.edem@abuad.edu.ng. https://orcid.
org/0000-0001-5921-5311
Blessing Uyo Nathaniel, Kate Eberechukwu Nebo and Azeez Olakunle
Ishola, Department of Anatomy, College of Medicine and Health
Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria
Abiola Oluwatosin Obisesan and Elizabeth Toyin Akinluyi,
Department of Pharmacology and Therapeutics, College of Medicine
and Health Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State,
Nigeria
Ayodeji Augustine Olabiyi, Department of Medical Biochemistry,
College of Medicine and Health Sciences, Afe Babalola University,
Ado-Ekiti, Ekiti State, Nigeria
Drug Metabol Pers Ther 2021; aop
blood [3]. In addition to driving cellular glucose meta-
bolism, insulin is also involved in the conversion of fats or
proteins to produce energy [4]. There is a fine and delicate
balance that mediates the regulatory roles and processes of
insulin in the body [5]. If insulin levels are too low or high,
excessively high or low blood sugar can start to cause
symptoms. If a state of low or high blood sugar continues,
serious health problems might start to develop. Insulin
signalling is initiated through binding and activation of its
cell-surface receptor and initiates a cascade of phosphor-
ylation and dephosphorylation events, second-messenger
generation, and protein-protein interactions that result in
diverse metabolic events in almost every tissue [6].
Lactobacillus plantarum (L. plantarum), a member of
the genus Lactobacillus is a versatile bacterium found in a
variety of ecological niches, ranging from vegetable and
plant fermentations to the human gastrointestinal tract.
L. plantarum cells are rods with rounded ends, straight,
generally 0.9–1.2 μm wide and 3–8μm long, occurring
singly, in pairs or short chains. This bacterium is most
frequently found in the fermentation of plant-derived raw
materials, which include several industrial and artisan
food and feed fermentations, like maize, olives, and a
variety of vegetables [7, 8]. As a lactic acid bacterium
(LAB), L. plantarum is widely employed in industrial
fermentation and processing of raw foods and “generally
recognized as safe”(GRAS) and has qualified presumption
of safety (QPS) status in the food processing sector [9]. Both
animal and human studies have demonstrated that diet
can influence the composition and function of the gut
microbiome [10]. Other factors, including genetics; the
mode of delivery at birth [11], the method of infant feeding
[12] and the use of medications, especially antibiotics, also
contribute to the decomposition and function of the gut
microbiome [13]. Diet plays an important role in obesity, in
addition to other factors. For example, yoghurt, a fer-
mented dairy product containing a variety of probiotic
bacteria, is found to be associated with a reduction in
inflammation markers and weight loss [14]. Studies found
that regular yoghurt consumption is involved in energy
balance and/or energy homeostasis, which in turn controls
body weight and reduces the risk of the development of
type 2 diabetes [15].
Male reproduction, development, and maintenance of
male sexual characteristics are principally governed by the
hypothalamic-pituitary-testicular (HPT) axis [16]. The axis
is composed of the hypothalamic gonadotropin-releasing
hormone (GnRH), luteinizing hormone (LH), follicle-
stimulating hormone (FSH), and the gonadal steroids
[17]. GnRH is the central regulator of the male reproductive
hormonal cascade [18]. The HPT axis maintains a dynamic
equilibrium of serum levels of reproductive hormones
through a closed-loop feedback mechanism [19]. As a
metabolic syndrome, hyperinsulinemia among other
metabolic disorders have been associated with disruptions
in the HPT axis resulting in alterations in reproductive
hormone levels including testosterone and gonadotropins,
as well as a significant increase in erectile dysfunction [20].
Also, increased insulin resistance has been reported in men
with unexplained infertility [21]. Therefore, this study seeks
to assess the efficacy of naturally-sourced biological agents
(L. plantarum) against a metabolic syndrome, hyper-
insulinemia co-morbid with infertility in male mice.
Materials and methods
Animals
An equal number (40) of male and female BALB/C mice weighing
between 25 and 30 g were obtained from Afe Babalola University, Ado-
Ekiti (ABUAD), Animal House. They were housed in well-ventilated
cages, kept and maintained under laboratory condition of tempera-
ture, humidity and light. They were allowed to acclimatize for a period
of two weeks and fed with broiler finisher pelletized meal. The mice
were also given drinking water ad libitum using water bottles.
Experimental design
At the end acclimatization of the animals, the male mice were assigned
to five groups (A, B, C, D and E) and treated accordingly: group
A (control) received normal saline for 28 days; group B (hyper-
insulinemia) given a high-fat diet (HFD) for 28 days, and thereafter
administered streptozotocin (STZ) intraperitoneally at a dose of
60 mg/kg on day 22 [22]. Group C (therapeutic group) mice were first
given an HFD for 28 days, and thereafter administered STZ intraperi-
toneally at a dose of 60 mg/kg on day 22 and were given at of 0.25 mL
L. plantarum from day 29 to 42 (14 days) of the experiment (post-
treatment). Group D (co-administration group) received HFD for
28 days, and thereafter administered STZ intraperitoneally at a dose of
60 mg/kg on day 22 while receiving 0.25 mL of L. plantarum treatment
from day 15 to 42 (simultaneous treatment).Group E (Probiotics group)
received L. plantarum for 14 days at a dose of 0.25 mL orally, as pre-
viously reported by Zavisic et al. [23] while the female mice were given
pelletized standard diet.
Ethical consideration
The animals were used according to the guidelines of the National
Council for Animal Experiments Control (CONCEA) and following
the National Institute of Health Guide for Care and Use of Labora-
tory animals [24]. The College Research Ethics Committee of Afe
Babalola University approved this study with protocol number AB/
EC/19/10/51.
2Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
Preparation of high-fat diet (HFD)
The HFD was prepared with quality ingredients by ABUAD Feed Mill
(ABUAD Farms, Ado-Ekiti, Nigeria) with composition as outlined in
Table 1. Preparation modified from Han et al. [25].
Induction of hyperinsulinemia
Mice were fed HFD for four weeks, then they were fasted overnight and
injected intraperitoneally (i.p.) with STZ (Sigma-Aldrich, USA) at a
dose of 60 mg/kg, which were freshly dissolved in 0.1 mM citrate
buffer (pH 4.5) [26]. The other groups (A, D and E) received citrate
buffer and 5 g/kg glucose was given the next day [27].
Preparation and administration of Lactobacillus
plantarum
The L. plantarum precipitate was collected after incubation for 18 h in
de Man, Rogosa and Sharpe (MRS) broth at 37 °C and centrifuged at
4,000×gfor 10 min. The number of viable bacteria was adjusted to
8×109cfu/mL by colony counting and then stored as a protectant
at −80 °C. Before administration, the required samples were freshly
prepared by 10-fold dilution of the stored L. plantarum and orally
administrated to the mice at a volume of 0.25 mL/kg body weight [28],
while the control group was given pre-warmed saline [29].
Sexual behavioural assay
To assess changes in sexual behaviours, oestrous female mice were
paired with males from experimental groups as described by Hull and
Dominguez [30] and Zheng et al. [31] with slight modifications. Sexual
behaviours were monitored in a separate room for 1 h in a clear plastic
box (60 ×60 ×80 cm) and recorded by digital video recording. On the
day of the test, mice were habituated to the test arena for 10 min before
introducing the oestrous female. Female mice were induced to oes-
trous by using two intraperitoneal doses of 0.5 μg of cloprostenol,
three days apart, plus a single subcutaneous dose of 3 μgofproges-
terone coincidentally with the first injection of cloprostenol [32] before
the determination of copulatory behaviours. The following sexual pa-
rameters were monitored and recorded; mounting latency (the time in-
terval between introduction of the female to the first mount by the male)
and intromission latency (the interval from the time of introduction of
the female to the first intromission by the male). The mounting number
(the number of mounts without intromission from the time of intro-
duction of the female to the male) and intromission number (the
number of intromissions from the time of introduction of the female
until the end of the experiment), was also recorded. A mount was
scored as the male climbing and grabbing the female from behind with
both paws while intromission was designated as vaginal penetration
during mounting accompanied by pelvic thrusting, as previously re-
ported by Hattori et al. [33].
Euthanasia
At the end of the experiment, the mice were sacrificed. This was done
by anaesthetizing with 100 mg/kg of ketamine hydrochloride. Blood
was collected for biochemical analyses. Animals for immunohisto-
chemical studies were transcardially perfused with phosphate buffer
solution (PBD, pH of 7.4), and then with 10% formal saline. Animals
for biochemical studies were perfused with phosphate buffer solution
only. Thereafter, the whole brain and testes were collected and fixed in
10% formal saline and Bouin’s solution, respectively. Samples for
biochemical assays were preserved in phosphate buffered saline (PBS)
and immediately frozen.
Seminal analyses
Epididymal sperm preparation: At the end of the experiment, a sem-
inal analysis was performed as previously described [34]. Briefly, the
cauda epididymis was cut longitudinally with a pair of fine-pointed
scissors and compressing with forceps. The sperm was released by
mincing the cauda epididymis into pieces on the Petri dishes that
contained phosphate buffer saline for sperm characteristics analyses.
The spermatozoa were allowed to flow out from cauda epididymis into
the buffer. Then, the sperm suspensions were left at room temperature
for 10 min for the suspension to allow sperm to swim out of the lumen
of the cauda epididymis for sperm characteristics analysis. Then
sperm motility, normal morphology, viability and sperm count were
evaluated for each animal.
Sperm motility, vitality, morphology and count evaluation: The
motility of the sperm was evaluated directly after mincing in drops of
sperm suspension on a microscopic slide. The light microscope was set
at ×40 eyepiece magnification for viewing. Motility was expressed as
the percentages of progressive motility including Rapid (Grade a) and
Slow (Grade b) spermatozoa, non-progressive (Grade c) and immotile
(Grade d) spermatozoa [35]. Sperm vitality analysis was done based on
a report by Bjorndahl et al. [36], Aitiken et al. [37] and Parhizkar et al.
[34]. Briefly, on a clean glass slide, one drop of sperm suspension was
gently mixed with three drops of eosin using the sharp glass slide end.
After 30 s, one drop of nigrosin was mixed with the solution and a
smear was made. The smear was then air-dried and observed under
×200 magnification of imaging microscope. The sperm cells were
counted based on the degree of membrane permeability (when viewed
under the light microscope). The dead sperm showed pink colouration
of the head whereas the vitality sperm showed whitish or colourless
head. Furthermore, sperm morphology analysis used the same sperm
smear made for sperm viability analysis. This time, the sperm were
observed under ×400 magnification of imaging microscope to
Table :Composition of experimental high-fat diet.
Ingredient Content, g/kg diet
Soybeans oil .
Lard .
Milk powder .
Corn-starch .
Sucrose .
Powdered cellulose .
Groundnut cake .
Mineral mixture .
Vitamin mixture .
Total, g/kg ,
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 3
evaluate the morphology of the sperm head, neck and tail according to
the Wryobek and Bruce criteria as reported by (Oliveira et al. 2009).
The sperm cells were generally classified as normal or abnormal
without further characterized types of abnormality found on the sperm
[38].
Sperm count was determined using the Neubauer haemocy-
tometer under a light microscope. A coverslip was placed on the
haemocytometer before a drop with 10 μL of caudal epididymal sperm
solution [34], which the sperm concentration was diluted with 3%
formal saline (1:20), was loaded under the coverslip. The haemocy-
tometer was placed under the light microscope, allowed to stand for
5 min for sedimentation and viewed under ×400 magnification. Sperm
count was done by counting 4 ×4 squares (horizontally or vertically).
Sperm count was determined using the formula below [39]:
Spermcount =totalno.ofspermin5squares ×50,000
×100 (cells/mL)
Counting was only done for sperm heads that were found within
the square’s areas.
Hormonal assays
Serum testosterone, Follicle-stimulating hormone, Luteinizing hor-
mone and Gonadotropin-Releasing hormone levels were measured
using Enzyme-Linked Immunosorbent Assay (ELISA) kit. The testos-
terone Equine testosterone antigen (ELA) was based on the principle of
competitive binding between testosterone in the test specimen and
testosterone-horseradish peroxidase (HRP) conjugate for a constant
amount of anti-testosterone. In the incubation, anti-testosterone
coated well were incubated with 25 μL of testosterone standards,
control, samples, 100 μL testosterone-HRP conjugate reagent at room
temperature for 60 min. During the incubation, a fixed amount of
HRP-labelled. Testosterone hydrogen-filled molecular graph (HFG)
competes with the endogenous testosterone in the standard, sample,
or quality control serum for a fixed number of binding sites of the
specific testosterone antibody. Thus, the amount of testosterone
peroxidase conjugate immunologically bound to the well progres-
sively decreases as the concentration of testosterone in the specimen
increases. Unbound testosterone peroxidase conjugate is then
removed and the wells washed. Next, a solution of 3,3′,5,5′-Tetrame-
thylbenzidine (TMB) reagent was then added and incubated at room
temperature for 15 min, resulting in the development of blue colour.
The colour development was stopped with the addition of a stop
solution, and the absorbance was measured using spectrophotometer
at 450 nm. Also, for FSH/LH hormonal assay, sixteen 12 ×75 mm
disposable plastic test tubes were labelled for the standards. Begin-
ning with 17, and two tubes for each clinical sample. Blank reagent
(300 μL), standards (200 μL), controls (200 μL) and clinical samples
(200 μL) were added to the sample bottles. One hundred microlitre of
LH/FSH antiserum was added to the tubes and vortexed. The tubes
were incubated for 30 min at room temperature. One millilitre of
precipitating reagent was added to all sample tubes, vortexed, and
centrifuged at 1,500×gfor 15 min.
The supernatant was carefully decanted from all tubes, except 1
and 2 immediately after centrifuging, inverting the tubes gently to
avoid disturbing the precipitate. Then the supernatant was discarded
properly. Counting the radioactivity in the pellets and tubes 1 and 2 in a
gamma counter for 1 min to obtain at least 40,000 counts for (57Co)
and 75,000 counts for (1,251) in the total count tubes. The total counts
in tubes 1 and 2 depended upon the efficiency of the scintillation
counter in use and the age of the tracer. Moreover, the micro ELISA
plate provided in the kit was being pre-coated with GnRH. During
the reaction, GnRH in the sample or standard competes with a
fixed amount of GnRH on the solid phase supporter for sites on the
Biotinylated Detection Ab specific to GnRH. Excess conjugate and
unbound sample or standard were washed from the plate, and Avidin
conjugated to Horseradish Peroxidase (HRP) was added to each
microplate well and incubated. Then a TMB substrate solution was
added to each well. The enzyme-substrate reaction was being termi-
nated by the addition of stop solution and the colour change was
measured with the use of spectrophotometer at a wavelength of
450 ±2 nm. The concentration of GnRH in the samples was then
determined by comparing the optical density of the samples to the
standard curve.
Serum lipid profile assessment
Total cholesterol (TC), total triglyceride (TGD) and high-density lipo-
protein (HDL) in serum were determined by a colorimetric assay kit
purchased from Fortress Diagnostics Limited (United Kingdom).
Antioxidant measurement
The testes were carefully dissected, washed in ice-cold saline, weighed
and thereafter, homogenized in PBS (pH 7.4). The homogenate was
centrifuged for 10 min at 5,000×gto yield a pellet that was discarded,
and a low-speed supernatant was kept for subsequent analysis. The
supernatant so obtained from the homogenized testis was used to
assay for the malondialdehyde (MDA) produced [40], insulin level [41]
and glutathione (GSH) activity [42].
Immunohistochemical assessment
Fixed hypothalamic and testicular tissues were cut into 10 μm-thick
sections. Insulin-like growth factor-1 receptor (IGF-IR) expression
was determined by immunohistochemical staining using rabbit
polyclonal IGF-IR primary antibody (BIORBYT ORB159743; dilution
1:200). After antigen retrieval with citrate buffer, endogenous
peroxidase activity was blocked by incubation in 3% hydrogen
peroxide. Nonspecific binding sites were blocked with goat serum
(Elabscience®E-IR-R111). HRP rabbit anti-goat HRP (BIORBYT
ORB216204, dilution 1:500) was applied as a secondary antibody.
Staining was performed with 3,3′-Diaminobenzidine (DAB), and
sections were counterstained with haematoxylin. Sections were
photographed using an OPTU-EDU light microscope.
Statistical analysis
Statistical analyses were done using one-way ANOVA (analysis of
variance), differences between groups were evaluated by the post-hoc
Tukey test with the aid of the GraphPad Prism Software (Graph Pad
Software Inc., CA, USA). The outcome of statistical analyses was
represented in graphs and bar charts with error bar representing the
mean ±SEM (standard error of the mean). Significant level was set at
p<0.05, p<0.01, p<0.001.
4Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
Results
Effect of L. plantarum on body weight
following HFD consumption and
streptozotocin exposure
Linear regression analysis shows significant changes in
body weight throughout the course of the experiment
across the groups with p-values 0.0001 (for control group),
0.0008 (for HFD +STZ group), 0.0472 (for therapeutic
[THRP] group), <0.0001 (for co-administration group type 1
[CO-AD]), and <0.0001 (for probiotics [PRBT] group).
Comparison between groups by one-way ANOVA followed
by the Tukey’s multi comparison as a post-hoc test showed
a significant difference in body weight following exposure
to HFD +STZ when compared to the control group
(p<0.001). Treatment with L. plantarum produced a sig-
nificant change in body weight in the group exposed to
HFD +STZ and treated simultaneously with L. plantarum
(CO-AD) when compared to the group that was exposed to
HFT +STZ only (p<0.05) (Figure 1).
Sexual behavioural indices
Effect of L. plantarum on intromission latency following
HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s Multiple Comparison showed a significant
increase in intromission latency following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.001), and in the
CO-AD group (p<0.01) significantly reduced intromission
latency compared to the HFD +STZ group (Figure 2A).
Effect of L. plantarum on intromission number following
HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
increase in intromission frequency following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to control group (p<0.001) significantly
increased intromission number when compared to the
HFD +STZ group (Figure 2B).
Effect of L. plantarum on mount latency following HFD
consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA
followed by the Tukey’s multiple comparison showed
a significant increase in Mount latency following
exposure to a high-fat diet and streptozotocin in the
HFD +STZ group when compared to control group
(p<0.001). Treatment with L. plantarum in the THRP group
(p<0.01), and in the CO-AD group (p<0.01) significantly
reduced mount latency compared to the HFD +STZ group
(Figure 2C).
Effect of L. plantarum on mounting number following HFD
consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparison showed a significant
decrease in Mount number following exposure to a high-fat
diet and streptozotocin in the HFD +STZ group when
compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.01), significantly
increased mount number compared to the HFD +STZ group
(Figure 2D).
Figure 1: Graph showing body weight changes following exposure
to HFD +STZ and treatment with Lactobacillus plantarum.
CTRL = control group; HFD +STZ = hyperinsulinemia group;
THRP = therapeutic group; CO-AD = co-administration group;
PRBT = probiotics only group. Comparison between groups by one-
way ANOVA followed by the Tukey’s multi comparison as a post-hoc
test, shows a significant difference in body weight following
exposure to HFD +STZ, when compared to the control group
(p<0.001). Treatment with L. plantarum produced a significant
change in body weight in the group exposed to HFD +STZ and
treated simultaneously with L. plantarum (CO-AD), when compared
to the group that was exposed to HFT +STZ only (p<0.05).
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 5
Seminal parameters
Effect of L. plantarum on sperm count following HFD
consumption and streptozotocin exposure
Comparison between groups byone-wayANOVAfollowedby
the Tukey’s multiple comparisons showed a significant
decrease in sperm count following exposure to a high-fat diet
and streptozotocin in the HFD +STZ group when compared to
control group (p<0.001). Treatment with L. plantarum in the
THRP group (p<0.05) significantly increased sperm count
compared to the HFD +STZ group (Figure 3A).
Effect of L. plantarum on sperm motility following HFD
consumption and streptozotocin exposure
Comparison between groups byone-wayANOVAfollowedby
the Tukey’s multiple comparisons showed a significant
decrease in sperm motility following exposure to a high-fat
diet and streptozotocin in the HFD +STZ group when
compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.01) significantly
increased sperm motility compared to the HFD +STZ group
(Figure 3B).
Effect of L. plantarum on sperm viability following HFD
consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA fol-
lowed by the Tukey’s multiple comparisons showed a
significant decrease in sperm viability following expo-
sure to a high-fat diet and streptozotocin in the
HFD +STZ group when compared to control group
(p<0.001). Treatment with L. plantarum in the THRP
group (p<0.01), and in the CO-AD group (p<0.05)
significantly increased sperm viability compared to the
HFD +STZ group (Figure 3C).
Effect of L. plantarum on sperm morphology following
HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in sperm morphology following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to the control group (p<0.05). Treatment
with L. plantarum produced a change in sperm
morphology, though no statistically significant when
compared with the HFD = STZ group (Figure 3D).
Figure 2: Bar chart showing intromission
latency (A), intromission number (B),
mounting latency (C) and mounting
frequency (D) changes following exposure
to HFD +STZ and treatment with
Lactobacillus plantarum.
CTRL = control group;
HFD +STZ = hyperinsulinemia group;
THRP = therapeutic group;
CO-AD = co-administration group;
PRBT = probiotics only group. Data are
expressed as mean ±SEM. p<0.05 was
considered to represent a significant
difference (ANOVA followed by post-hoc
Tukey, n = 8; ns denotes not significant,
*denotes p<0.05, **denotes p<0.01 and
***denotes p<0.001).
6Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
Biochemical assessments
Effect of L. plantarum on blood lipid profile following HFD
consumption and streptozotocin exposure
Effect of L. plantarum on blood total cholesterol level
following HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
increase in Blood Total Cholesterol following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.001), and in the
CO-AD group (p<0.01) significantly reduced Blood Total
Cholesterol compared to the HFD +STZ group (Figure 4A).
Effect of L. plantarum on blood high-density lipoprotein
(HDL) level following HFD consumption and streptozotocin
exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in Blood High-Density Lipoprotein following
exposure to a high-fat diet and streptozotocin in the
HFD +STZ group when compared to control group
(p<0.001). Treatment with L. plantarum in the THRP group
(p<0.001), and in the CO-AD group (p<0.001) significantly
increased blood HDL compared to the HFD +STZ group
(Figure 4B).
Effect of L. plantarum on blood triglyceride level following
HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
increase in Blood Triglycerides following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.001), and in the
CO-AD group (p<0.001) significantly reduced Blood Tri-
glycerides compared to the HFD +STZ group (Figure 4C).
Effect of L. plantarum on blood insulin level following HFD
consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
Figure 3: Bar chartshowing sperm count(A), motility (B), viability (C) and morphology (D) changesfollowing exposure to HFD +STZ and treatment
with Lactobacillus plantarum.
CTRL = control group; HFD +STZ = hyperinsulinemia group; THRP = therapeutic group; CO-AD = co-administration group; PRBT = probiotics
only group. Data are expressed as mean ±SEM. p<0.05 was considered to represent a significant difference (ANOVA followed by post-hoc
Tukey, n=8; ns denotes not significant, *denotes p<0.05, **denotes p<0.01 and ***denotes p<0.001).
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 7
increase in Blood Insulin following exposure to a high-fat
diet and streptozotocin in the HFD +STZ group when
compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.001), and in the
CO-AD group (p<0.05) significantly reduced Blood Insulin
Level compared to the HFD +STZ group (Figure 4D).
Effect of L. plantarum on reproductive hormone levels
following HFD consumption and streptozotocin exposure
Effect of L. plantarum on serum follicle-stimulating hor-
mone (FSH) level following HFD consumption and strep-
tozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in Blood Follicle-Stimulating hormone following
exposure to a high-fat diet and streptozotocin in the
HFD +STZ group when compared to control group
(p<0.01). Treatment with L. plantarum in the THRP group
(p<0.05), significantly increased FSH compared to the
HFD +STZ group (Figure 5A).
Effect of L. plantarum on serum testosterone level
following HFD consumption and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decreased Testosterone following exposure to a high-fat
diet and streptozotocin in the HFD +STZ group when
compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.05), and significantly
increased Testosterone compared to the HFD +STZ group
(Figure 5B).
Effect of L. plantarum on serum gonadotrophin-releasing
hormone level (GnRH) level following HFD consumption
and streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in GnRH following exposure to a high-fat diet and
streptozotocin in the HFD +STZ group when compared to
control group (p<0.01). Treatment with L. plantarum in the
THRP group (p<0.05), a significant increase in GnRH
compared to the HFD +STZ group (Figure 5C).
Figure 4: Bar chart showing blood total cholesterol, HDL, insulin and triglycerides changes following exposure to HFD +STZ and treatment with
Lactobacillus plantarum.
CTRL = control group; HFD +STZ = hyperinsulinemia group; THRP = therapeutic group; CO-AD = co-administration group; PRBT = probiotics only
group. Data are expressed as mean ±SEM. p<0.05 was considered to represent a significant difference (ANOVAfollowed by post-hoc Tukey, n=8;
ns denotes not significant, *denotes p<0.05, **denotes p<0.01 and ***denotes p<0.001).
8Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
Effect of L. plantarum on serum luteinizing hormone (LH)
level following HFD consumption and streptozotocin
exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in Luteinizing hormone following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to the control group (p<0.001). Treatment
with L. plantarum in the THRP group (p<0.05), significantly
increased Luteinizing hormone compared to the HFD +STZ
group (Figure 5D).
Testicular oxidative stress markers
Effect of L. plantarum on testicular glutathione (GSH)
level following HFD consumption and streptozotocin
exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
decrease in Testicular Glutathione following exposure to a
high-fat diet and streptozotocin in the HFD +STZ group
when compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.001), and in the
CO-AD group (p<0.001) significantly increased Glutathione
compared to the HFD +STZ group (Figure 6A).
Effect of L. plantarum on testicular malondialdehyde
(MDA) level following HFD consumption and
streptozotocin exposure
Comparison between groups by one-way ANOVA followed
by the Tukey’s multiple comparisons showed a significant
increase in Testicular MDA following exposure to a high-fat
diet and streptozotocin in the HFD +STZ group when
compared to control group (p<0.001). Treatment with
L. plantarum in the THRP group (p<0.01), and in the CO-AD
group (p<0.01) significantly reduced Testicular MDA
compared to the HFD +STZ group (Figure 6B).
Immunohistochemical assessments
Immunoreactivity for insulin growth-like factor-1 (IGF-1)
receptor in the hypothalamus and testes
In the hypothalamus, Group A shows increased immuno-
reactivity for IGF-1 receptors, while there is decreased IGF-1
immunoreactivity in Group B. Groups C and D reveal
improved immunoreactivity for IGF-1 receptor (Figure 7).
Figure 5: Bar chart showing hormonal (FSH,
A; Testosterone, B; GnRH, C and LH, D) level
following exposure to HFD +STZ and
treatment with Lactobacillus plantarum.
CTRL = control group; HFD +STZ =
hyperinsulinemia group; THRP = therapeutic
group; CO-AD = co-administration group;
PRBT = probiotics only group. Data are
expressed as mean ±SEM. p<0.05 was
considered to represent a significant
difference (ANOVA followed by post-hoc
Tukey, n=8; ns denotes not significant,
*denotes p<0.05, **denotes p<0.01 and
***denotes p<0.001).
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 9
Also, as represented in Figure 8 evaluating for immuno-
reactivity for IGF-1 in the testes, Group A shows increased
immunoreactivity for IGF-1 receptors especially in the
spermatids (Spt), while there is decreased IGF-1 immuno-
reactivity and significant loss of spermatids with evident
increase in seminiferous tubule (ST) luminal diameter
(LuM) in Group B. Groups C and D reveal improved
immunoreactivity of IGF-1 receptors (Figure 8).
Discussion
Type 2 diabetes, strongly associated with hyperinsulinemia
has been reported to increase body weight and food intake
in mice, due to the leptin receptor deficiency and ghrelin
deficiency [43]. In the present study, a combination of
HFD +STZ was employed to model hyperinsulinemia in
mice. An increase in the average body weight was observed
Figure 6: Bar chart showing testicular GSH
(A) and MDA (B) levels following exposure to
HFD +STZ and treatment with Lactobacillus
plantarum.
CTRL = control group; HFD +STZ =
hyperinsulinemia group;
THRP = therapeutic group; CO-AD = co-
administration group; PRBT = probiotics
only group. Data are expressed as
mean ±SEM. p<0.05 was considered to
represent a significant difference (ANOVA
followed by post-hoc Tukey, n=8; ns
denotes not significant, *denotes p<0.05,
**denotes p<0.01 and ***denotes p<0.001).
Figure 7: Photomicrographs showing immunoreactivity for IGF-1 in the hypothalamus.
(A) control group; (B) HFD +STZ (hyperinsulinemia group); (C) therapeutic group; (D) co-administration group; (E) probiotics only group. Scale
bar: 42 µm.
10 Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
during the first four weeks in the therapeutic group and
HFD +STZ group when compared to the control. The in-
crease in body weight is associated with an increase in
ghrelin level which causes motivation towards food [43].
During the third and fourth week, decrease in body weight
was observed in the CO-AD group when compared to the
control. This finding agrees with the report of Takemura
et al. [44] where the effects of L. plantarum strain No. 14
(LP14), was reported to decrease body fat percentage in
healthy volunteers.
Type 2 diabetes has been documented to reduce
endogenous alpha-melanocyte-stimulating hormone lead-
ing to low sexual function or motivation [45]. The sexual
behavioural indices in the present study showed a signifi-
cant decrease in sexual motivation as a result of a significant
decrease in mounting frequency (MF) and intromission fre-
quency (IF) of the HFD +STZ group and a significant in-
crease in mounting latency (ML) and intromission latency
(IL) in the HFD +STZ group compared to the control group.
Hormonal control or balance have contributed to improved
sexual function in several studies reported [46]. Testos-
terone treatment or replacement is one of the management
options put in place for improved sexual function. Also,
FSH/LH is triggered with improved or increased testosterone
level. There is a rise in the call for nutritional modulation in
the management of metabolic disorders like diabetes and its
complications, including infertility. Probiotics treatments
have been employed in the restoration of testosterone bal-
ance following oxidative and inflammatory damages [47].
Treatment with L. plantarum produced a significant reduc-
tion in intromission latency, mount latency and mount
number when compared to HFD +STZ group. Though the
mechanisms underlying sexual behaviours are not fully
understood, it can be hypothesized that dopamine plays an
important role in the neural processes of sexual behaviours
especially for motor functions and general arousal [48, 49].
Administration of probiotics like L. plantarum has been
shown to improve dopamine activities as well as prevent
dopamine loss [50], therefore this study suggests that the
observed improvement in sexual behaviour following
administration of L. plantarum to hyperinsulinemic mice is
via the modulation of dopamine signalling pathway.
Diabetes mellitus may affect male reproductive func-
tions at multiple levels including its detrimental effects on
Figure 8: Photomicrographs showing immunoreactivity for IGF-1 in the testis.
(A) control group; (B) HFD +STZ (hyperinsulinemia group); (C) therapeutic group; (D) co-administration group; (E) probiotics only group. Scale
bar: 42 µm.
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 11
endocrine control of spermatogenesis and/or by impairing
erection and ejaculation [51]. Ricci et al. [52] found that
insulin-dependent diabetes is accompanied by reduced
semen volume and decreased vitality and motility of the
spermatozoa, with no change in seminal viscosity. Another
study revealed that high level of blood sugar may affect
sperm quality and therefore decrease male fertility poten-
tials [53]. Moreover, some confirmations have indicated
higher rates of infertility in diabetic men and poor repro-
ductive outcomes in comparison with healthy men [54]. In
this study, sperm quality was observed to be significantly
reduced in the HFD +STZ group when compared with the
control group whereas treatment with L. plantarum in the
therapeutic group showed a significant increase in sperm
motility, count and viability when compared to the
HFD +STZ group. Co-administration of L. plantarum
simultaneously with the induction of hyperinsulinemia in
the CO-AD group showed a significant increase in sperm
vitality when compared to the HFD +STZ group. However,
no significant difference in sperm morphology was
observed in the two treatment groups (THRP and CO-AD)
when compared to the HFD +STZ group. We can therefore
suggest that L. plantarum improves sperm quality by
increasing its vitality with no altered morphology.
Hyperlipidaemia (usually elevated serum levels of TC,
TGD and low-density lipoprotein [LDL], accompanied by a
reduced HDL level) is strongly associated with cardiovas-
cular diseases (CVDs) [55]. An increase was observed in the
TC, LDL, TGD and insulin levels in the HFD +STZ group
when compared to the control group. Administration of
L. plantarum had a significant effect on the TC and TGD
parameters by lowering the levels of TC and TGD through
the mechanism of take-up and assimilate cholesterol for
stabilization of their cell membrane and binding choles-
terol walls with the probiotic (L. plantarum). The HDL
levels in the control and probiotic groups significantly rose
when compared to that in the HFD +STZ group. The THRP
and CO-AD groups following treatment with L. plantarum
had a significant increase in the HDL levels when
compared to HFD +STZ group. Treatment with L. planta-
rum significantly lowered blood TC, TGD and insulin levels
in the THRP and CO-AD when compared to the diabetes
group. This report agrees with Li et al. [56] where
cholesterol-lowering effect of L. plantarum was evaluated.
Also, LDL was found to be the most dangerous factor
among serum lipids owing to increasing penetration of
oxidized LDL into arterial walls and forming atheroscle-
rotic plaque lesions [55, 57]. Decreasing serum TC and LDL
levels are effective for reducing the risk of atherosclerosis
[58]. Furthermore, the hormonal study showed a signifi-
cant reduction in the serum FSH, LH, GnRH and
Testosterone levels in the HFD +STZ group when compared
to the control. Treatment with the L. plantarum in the
therapeutic group significantly increased all the hormones
levels. However, no significant changes were observed in
the CO-AD group, because the amount of probiotic absor-
bed in the gut while exposed to HFD +STZ was probably
not enough to reduce the glucose consumption, thus
increasing the amount of glucose absorbed into the blood
[59].
The male reproductive system, and in particular the
testicular tissue are susceptible to oxidative stress [60].
Oxidative stress is an important factor in male infertility
because of a very high rate of cell division and mitochon-
drial oxygen consumption, as well as comparably higher
levels of unsaturated fatty acids in testicular tissue than in
other tissues [61]. Moreover, weakness of the testicular
artery results in low oxygen pressure; hence there is a
severe cell competition for oxygen. Oxidative stress plays a
major role in the progression and pathogenesis of meta-
bolic disorders including diabetes, obesity and hyper-
insulinemia as observed in the HFD +STZ-induced
diabetes and hyperinsulinemic mice. Among various
functional foods with an antioxidant effect, probiotics like
L. plantarum have been reported to repress oxidative stress
[62]. In the diabetic condition, oxidative stress impairs
glucose uptake in muscle and fat [63] and decreases insulin
secretion from pancreatic βcells [64]. It was observed that
diabetes induction in mice could significantly induce
oxidative stress in the testis by increasing the MDA levels
and decreasing the levels of GSH [65]. In the present study,
we hypothesized that probiotic treatment could prevent
and ameliorate testicular oxidative stress. Hyper-
insulinemic mice treated with L. plantarum showed
improved GSH levels which is similar to what was reported
by Yigitturk et al. [66]. Testicular MDA level was signifi-
cantly increased in the HFD +STZ-treated mice when
compared to the control mice. Meanwhile, the antioxidant
activity of GSH was significantly reduced in the testes of the
HFD +STZ-treated animals when compared with the con-
trol group. Therefore, probiotic treatment attenuated
testicular oxidative effects by significantly reducing the
MDA level and increasing the GSH level in the testes of
THRP and CO-AD groups, suggesting that L. plantarum is
capable of ameliorating testicular oxidative stress in
HFD +STZ-induced diabetes and hyperinsulinemia.
Insulin signalling is initiated through binding and
activation of its cell-surface receptor (Insulin-like growth
factor-1 receptor, IGF-1R) and initiates a cascade of phos-
phorylation and dephosphorylation events [67]. The
phosphorylation of IRS (Insulin receptor substrate) pro-
teins on tyrosine residues activate insulin signalling which
12 Edem et al.: Lactobacillus plantarum mitigates reproductive deficits
in turn stimulates glucose transport into the target cell,
through the downstream activation of IGF-1 [68]. Insulin
acts as a growth factor and stimulates cell growth, differ-
entiation and survival [69]. Insulin has been reported to
execute two important functions in the hypothalamus;
suppression of food intake and the improvement of glucose
metabolism. These functions exert advantageous effects on
obesity and diabetes, respectively [70]. Obesity and over-
consumption of saturated fats lead to a condition known as
hypothalamic insulin resistance which leads to a disrup-
tion of the functions performed by insulin in the hypo-
thalamus. Several studies have tried to explain the specific
part of the insulin signalling pathway that is blocked in the
hypothalamus. Several of these studies have reported a
decrease in tyrosine phosphorylation of IR and IRS [71] due
to increase in two tyrosine phosphatases, tyrosine-protein
phosphatase non-receptor type 1 (PTB-1B) [72] and T-cell
protein tyrosine phosphatase (TCPTP) [73] to be the main
cause of altered insulin signalling in the hypothalamus in
obese patients. However, only long term HFD feeding has
been shown to increase both phosphatases [70]. Further-
more, alteration in the level of activation of IGF-1R has been
associated with type 2 diabetes [74].
Immunohistochemical assessment in the present study
has shown a decrease in immunoreactivity for the IGF-1 re-
ceptor in the hypothalamus of the HFD +STZ group,
compared to the control group. Treatment with L. plantarum
produced an improvement in the expression of IGF-1 re-
ceptors in the hypothalamus of the THRP and CO-AD
groups. This shows that long term treatment with L. planta-
rum resolves the damage done in the IGF-1 receptor by
reactivating insulin signalling pathway in the hypothalamic
regions of the brain [75, 76]. Insulin/IGF-1 receptor plays a
role in testicular development, sexual performance and
testicular function. IGF-1 receptor has been known to
interact with the hypothalamic-pituitary-testicular axis by
directly and indirectly influencing reproductive function
through regulating gonadotrophin-releasing hormone [77].
However, it is also possible that the adverse effects of dia-
betes on male fertility may be due to abnormal insulin sig-
nalling in the testis. In diabetes, chronic hyperglycaemia
causes oxidative stress by generating free radicals in cells of
the testis including spermatogenic, Sertoli, Leydig and
endothelial cells [78]. Destruction of these cells could bring
about male infertility [79]. Due to the involvement of
oxidativestress in chronic hyperglycaemia-induced damage
to the reproductive structures as it occurs in diabetes,
treatment or management with antioxidants stand a high
chance of mitigating such damage [80]. The present study
has demonstrated that L. plantarum effectively enhanced
sexual function, increased motility and sperm count [56].
Additionally, immunohistochemical evaluation in the pre-
sent study reveals that immunoreactivity for the IGF-1 re-
ceptor in the control testes was increased, especially in the
spermatids. In the HFD +STZ group, a decreased expression
of IGF-1 receptor with significant loss of spermatids due to
the disruption of hormone homeostasis of the
hypothalamic-pituitary-testicular axis was observed. This
resulted in testicular morphological changes, like Leydig
cells shrinking, with an evident increase in the seminiferous
tubules lumina.
Conclusions
Taken together, the findings of the present study demon-
strate that oral administration of L. plantarum was able to
mitigate hyperinsulinemia-induced male sexual and
reproductive dysfunctions by modulating antioxidant
imbalance, lipid and insulin metabolism in the
hypothalamic-pituitary-testicular axis of mice. These
findings, therefore, support the therapeutic application of
probiotics in the management of diabetes/hyper-
insulinemia and its co-morbidities, like infertility in males.
Acknowldgements: The authors acknowledge the support
in form of facilities and materials provided for use in this
stud by Afe Babalola University Ado-Ekiti.
Research funding: None declared.
Author contributions: EEE: Conceptualization, methodology,
supervision and editing. BUN: Project administration,
resources and writing of the original draft. KEN: Project
administration and data curation. AOO: Validation, project
administration and resources. AAO: Validation, reviewing and
editing. AOI and ETA: Resources, data curation and formal
analyses. All authors reviewed the manuscript final draft
before submission. All authors have accepted responsibility for
the entire content of this manuscript and approved its
submission.
Competing interests: Not applicable.
Informed consent: Informed consent was obtained from all
individuals included in this study.
Ethical approval: The animals were used according to the
guidelines of the National Council for Animal Experiments
Control (CONCEA) and following the National Institute of
Health Guide for Care and Use of Laboratory animals [24].
The College Research Ethics Committee of Afe Babalola
University approved this study with protocol number
AB/EC/19/10/51.
Edem et al.: Lactobacillus plantarum mitigates reproductive deficits 13
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