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Received: 5 August 2018 Revised: 27 September 2018 Accepted: 14 October 2018 DOI: 10.1111/and.13196
ORIGINAL ARTICLE
LY294002, a PI3K pathway inhibitor, prevents leptin‐induced adverse effects on spermatozoa in Sprague‐Dawley rats Amir Hafidz Md Mokhtar1 | Ifrah Alam Malik1 | Noor Azean Anis Abd Aziz1 Fayez A. Almabhouh1
| Damayanthi Durairajanayagam1
1 Faculty of Medicine, Universiti Teknologi MARA, Sg Buloh, Selangor, Malaysia
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| Harbindar Jeet Singh1,2
Abstract
2 I‐PPerForM , Faculty of Medicine, Universiti Teknologi MARA, Sg Buloh, Selangor, Malaysia
This study examined the effects of PI3K and AMPK signalling pathway inhibitors on
Correspondence Harbindar Jeet Singh, Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia. Email:
[email protected]
hibitor)‐, and leptin+LY294002 (PI3K inhibitor)‐treated groups with six rats per group.
Funding information Fundamental Research Grant Scheme, [600RMI/FRGS 5/3 (0011/2016)]; Ministry of Higher Education, Malaysia
14 days. Controls received 0.1 ml of normal saline. Upon completion, sperm count,
leptin‐induced adverse effects on rat spermatozoa. Sprague‐Dawley rats, aged 14–16 weeks, were randomised into control, leptin‐, leptin + dorsomorphin (AMPK in‐ Leptin was given once daily for 14 days via the intraperitoneal (i.p.) route at a dose of 60 µg kg−1 body weight. Rats in the leptin and inhibitor‐treated groups received concur‐ rently either dorsomorphin (5 mg kg−1 day−1) or LY294002 (1.2 mg kg−1 day−1) i.p. for sperm morphology, seminiferous tubular epithelial height (STEH), seminiferous tubular diameter (STD), 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) and phospho‐Akt/total Akt ratio were estimated. Data were analysed using ANOVA. Sperm count, STEH and STD were significantly lower, while the percentage of spermatozoa with abnormal morphol‐ ogy and the level of 8‐OHdG were significantly higher in rats treated with leptin and leptin + dorsomorphin when compared to those in controls and LY294002‐treated rats. Testicular phospho‐Akt/total Akt ratio was significantly higher in leptin and lep‐ tin + LY294002‐treated rats. In conclusion, LY294002 prevents leptin‐induced changes in rat sperm parameters, suggesting the potential role of the PI3K signalling pathway in the adverse effects of leptin on sperm parameters. KEYWORDS
dorsomorphin, leptin, LY294002, PI3K, sperm parameters
1 | I NTRO D U C TI O N
the fraction of spermatozoa with abnormal morphology in Sprague‐ Dawley rats (Almabhouh et al., 2017, 2015; Haron, D’Souza, Jaafar,
Leptin, a 16 kDa non‐glycosylated peptide hormone, is produced
Zakaria, & Singh, 2010). It also increased the levels of FSH and LH
and secreted by white adipose tissue. It is involved in the regulation
but had no effect on testosterone (Abbasihormozi et al., 2013;
of food intake and body weight, inflammation, immunity, haemato‐
Haron et al., 2010). More recently, exogenous leptin treatment has
poiesis, sexual maturation and reproduction (Kiess, Blum, & Aubert,
been reported to decrease the replacement of histone by protamine
1998; Tena‐Sempere & Barreiro, 2002). Leptin restores fertility in
in spermatozoa of Sprague‐Dawley rats (Almabhouh & Singh, 2018).
leptin‐deficient ob/ob mice, indicating that leptin is required for
Leptin‐induced oxidative stress has been implicated in these
normal reproductive function (Barash et al., 1996; Mounzih, Lu, &
adverse effects. Leptin administration to rats has been shown to
Chehab, 1997). However, recent reports indicate significant adverse
increase free radical production, increase sperm DNA fragmenta‐
effects of leptin on rat spermatozoa. Daily leptin treatment for a du‐
tion and apoptosis in the seminiferous tubules (Abbasihormozi et
ration of 1–6 weeks decreased sperm concentration and increased
al., 2013; Almabhouh et al., 2017), and increase the levels of sperm
Andrologia. 2018;e13196. https://doi.org/10.1111/and.13196
wileyonlinelibrary.com/journal/and
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8‐hydroxy‐2‐deoxyguanosine (8‐OHdG), a DNA marker of oxida‐
(Recombinant Rat Leptin; Purity >95%, Biovision, USA). In the
tive stress (Almabhouh et al., 2017, 2015). Besides this, concurrent
leptin‐ and inhibitor‐treated groups, the animals were given ei‐
melatonin administration has been shown to prevent leptin‐induced
ther dorsomorphin (5 mg kg−1 day−1) or LY294002 (PI3K inhibitor;
adverse effects on spermatozoa in the rat (Almabhouh et al., 2017).
1.2 mg kg−1 day−1) i.p. together with leptin for 14 days. Control
The signalling pathway responsible for these leptin‐induced adverse
rats received 0.1 ml of normal saline i.p. for 14 days. Body weight
effects remains unclear. Intracellular signalling pathways that are
of both the control and experimental animals was recorded
activated by leptin include the phosphatidylinositol 3‐kinase (PI3K),
weekly. The doses of leptin and dorsomorphin used in this study
mitogen‐activated protein kinase (MAPK) and mammalian target of
were according to Almabhouh et al., 2015, and Pachori et al., 2010,
rapamycin (mTOR), adenosine monophosphate‐activated protein ki‐
respectively. The dose of LY294002 used was according to Shan
nase (AMPK), and JAK‐STAT pathways (Fruhbeck, 2006; Kwon, Kim,
et al., 2008. The duration of treatment was based on Haron et al.,
& Kim, 2016). Among these signalling pathways, those that are associ‐
2010.
ated with oxidative stress include the AMPK, PI3K, MAPK and mTOR pathways. Interestingly, microarray analysis of testicular tissue from leptin‐
2.2 | Sperm collection
treated rats in our laboratory showed a significant upregulation of
Upon completion of treatment, the animals were euthanised by de‐
the gene expression of proteins involved in the PI3K pathway, such
capitation using a small animal guillotine. The testes and epididymis
as AKT, protein kinase c (PKC), 3‐phosphoinositide‐dependent ki‐
were excised and weighed. The epididymis was minced in 2 ml of
nase‐1 (PDK1) and phosphodiesterase (PDE; fold change and p value;
0.9% saline and then filtered through a nylon mesh to prepare an
2.14 p = 0.0088, 2.18 p = 0.0087, 5.36 p = 0.0066, 2.39 p = 0.0163
epididymal suspension for analysis (Almabhouh et al., 2015; Haron
respectively), but no changes were noted in AMP‐activated protein
et al., 2010). The right testis was immediately placed in 10% (v/v)
kinase genes (unpublished data from a study that was published
buffered formalin for histopathology study.
recently, Almabhouh et al., 2017). In order to confirm these earlier microarray findings, the present study examined the involvement of PI3K and AMPK signalling pathways in leptin‐induced changes in
2.3 | Sperm count and morphology
sperm parameters following treatment with LY294002 (a commonly
Sperm count and sperm morphology were determined using the
used PI3K inhibitor; Shan et al., 2008) and dorsomorphin (an AMPK
Makler chamber. The epididymal suspension was first mixed well,
pathway inhibitor; Pachori et al., 2010).
and a small droplet was placed in the centre of the Makler cham‐ ber (Sefi Medical Instruments LTD, Haifa) and covered with a glass
2 | M ATE R I A L S A N D M E TH O DS
cover. The droplet was allowed to spread over the entire area of the disc. The total number of spermatozoa (normal and abnormal) in 10 squares was counted, representing the sperm concentration
2.1 | Experimental animals
in million per ml. This step was then repeated, and the average of two counts was determined. The concentration was expressed
Sexually‐matured, male Sprague‐Dawley rats, aged between 14
as million per ml. The number of abnormal spermatozoa in the
and 16 weeks and weighing between 400 and 450 g, were ac‐
same 10 squares was recorded, and the fraction of spermato‐
quired from the Laboratory Animal Care Unit (LACU), Universiti
zoa with abnormal morphology was calculated as a percentage.
Teknologi MARA. The choice of age of the rats was based on our
Spermatozoa with abnormal morphology include those that are
very early studies where we found that the sperm concentra‐
headless, with cephalo‐caudal defects, coiled tailed, hookless,
tion in the rats in our laboratory reaches its maximum concen‐
broken tailed, double‐headed and double‐tailed spermatozoa
tration by 14–16 weeks of age. Over the duration of the study
(Narayana, D’Souza, & Rao, 2002).
period, the rats were housed individually in metabolic cages at room temperature (22–24°C) and with a 12:12‐hr light/dark cycle. Throughout the experimental period, the animals had access ad li‐
2.4 | Histological evaluation of testis
bitum to tap water and commercial rat feed (Rodent Diet Specialty
Testicular tissues were first fixed in 10% (v/v) buffered formalin,
Feeds, Glen Forrest, Western Australia). The experimental proto‐
then embedded in paraffin wax and later cut into thin sections
col used in this study was approved by the Research Committee
(5 µm thickness) with a microtome. These were then stained with
on the Ethical Use of Animals (UITM CARE 163/2017), Universiti
haematoxylin and eosin (H&E). Seminiferous tubular diameter
Teknologi MARA, Malaysia.
(STD) measurement was taken from one basement membrane to
The rats were randomised into four groups, that is control,
the next basement membrane, while the seminiferous tubule epi‐
leptin‐, leptin + dorsomorphin (AMPK inhibitor)‐ and leptin +
thelial height (STEH) measurement was taken from the basement
LY294002 (PI3K inhibitor)‐treated groups, with each group con‐
membrane to the surface of the epithelium (Haron et al., 2010).
sisting of six rats. Leptin was given once daily for 14 days via the
Measurements were obtained from 10 seminiferous tubules from
intraperitoneal (i.p.) route at a dose of 60 µg/kg body weight
each sample and then averaged.
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of variance (ANOVA) was therefore used to analyse the data fol‐
2.5 | Quantitative measurement of 8‐OHdG
lowed by post hoc analysis using the Tukey test. These tests were
Levels of 8‐OHdG were measured in testicular tissue using an ELISA
run using SPSS version 20 (IBM, NY, USA) software. The data are
kit (Elabscience, Houston, TX, USA). The testicular tissue was initially
expressed as mean ± SEM. A p
