Journal of Animal Reproduction and Biotechnology 2020; 35(1): 73-85
Published online March 31, 2020
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Changwon Yang1, Hyocheol Bae1, Gwonhwa Song1,* and Whasun Lim2,*
1Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
2Department of Food and Nutrition, College of Science and Technology, Kookmin University, Seoul 02707, Korea
A high-cholesterol diet can reduce male fertility. However, it is not known whether a high-cholesterol diet can regulate the expression of genes involved in sperm maturation and sperm fertilizing ability. Quercetin, a natural product, is known to have cytoprotective effects by regulating lipid metabolism in various cell types. This study aimed to confirm the expression of genes involved in sperm maturation in the testes of mice fed a high-cholesterol diet and to determine whether quercetin can reverse the genetic regulation of cholesterol. Mice were divided into groups fed a normal chow diet and a high-cholesterol diet. Mice fed the high-cholesterol diet were dose-dependently supplemented with quercetin for 6 weeks. Investigations using quantitative PCR and in situ hybridization revealed that the high-cholesterol diet alters the expression of genes associated with sperm maturation in the testes of mice, and this was reversed with the supplementation of quercetin. In addition, the high-cholesterol diet regulated the expression of genes related to lipid metabolism in the liver of mice. Under a highcholesterol diet, quercetin can improve male fertility by regulating the expression of genes involved in sperm maturation.
Keywords: cholesterol, lipid metabolism, quercetin, spermatogenesis, testis
Approximately 30% of cases of male infertility have no known cause (Cavallini, 2006). Several studies have shown that obesity-related diseases are associated with a decrease in male reproductive potential, which is evidenced by a decrease in sperm parameters and testosterone levels (Lotti et al., 2013; Leisegang et al., 2014). The testis is the organ responsible for fertility by producing sperm and it functions through the physiological action of various types of cells, including Leydig cells, Sertoli cells, germ cells, and peritubular cells (Majumdar and Bhattacharya, 2013). Cholesterol is a structural component that maintains the fluidity of cell membranes and plays an important role in regulating the physiological function of mammalian cells. Moreover, cholesterol is a precursor to steroid synthesis, which is essential for the production of normal sperm (Yokoyama, 2000). Cholesterol homeostasis is also disturbed by exposure to environmental toxins, which in turn disrupts spermatogenesis and the development of gonads (Samova et al., 2018). In rabbits, hypercholesterolemia damages Sertoli and Leydig cells in the testes and reduces sperm quality (Saez Lancellotti et al., 2010). Moreover, in male mice fed a high-cholesterol diet, the testes have been shown to have low steroidogenic enzymes and high oxidative stress (Wang et al., 2015). However, it is not known whether a cholesterol diet can affect the expression of genes related to sperm maturation and sperm fertilizing ability.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a polyphenolic flavonoid widely found in vegetables and fruits and has been shown to improve various health functions with anti-cancer, antioxidant, and anti-inflammatory effects. Quercetin also alters gene transcription and can affect signal transduction pathways that regulate cell proliferation (Brito et al., 2015). In addition, quercetin has been shown to reduce cholesterol synthesis and regulates lipid metabolism in several cell types, including pancreatic β-cells and glial cells (Carrasco-Pozo et al., 2016; Damiano et al., 2019). Moreover, quercetin is known to mitigate the harmful effects of various chemicals that damage the testes and cause reproductive toxicity in mammals (Izawa et al., 2008; Abdel Aziz et al., 2018). However, it is unclear whether quercetin can control genetic changes in the testes caused by a high-cholesterol diet.
In our previous studies, we found that high-cholesterol diets cause histological alterations in the male reproductive organs in rodents (Lim et al., 2015). In this study, we examined the change in sperm adhesion molecule 1 (
The experimental use of mice in this study was approved by the Animal Care and Use Committee of Korea University. Four-week-old male C57BL/6 mice were maintained for an adaptive period of 10 days with ad libitum diet in conditions of 12 h light and 12 h dark, according to the institutional guidelines for animal care. Afterward, a total of 48 mice were divided into six groups. Group 1 (n = 8) was fed a normal diet and Groups 2 through 6 (n = 8 per each group) were fed a high-cholesterol diet containing 3% cholesterol and 0.5% cholic acid. Groups 3, 4, and 5 were additionally fed 25, 50, and 100 mg/kg quercetin, respectively. Finasteride (10 mg/kg) was added to the feed of group 6. Each diet was provided for 6 weeks. The composition of normal and high-cholesterol diets is provided in Table 1.
Mice were euthanized after starving for 12 h. The testis and liver tissues were then stored in liquid nitrogen for RNA extraction and in 4% paraformaldehyde in phosphate buffered saline for tissue fixation.
Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA. AccuPower RT premix (Bioneer, Daejeon, Korea) was used to synthesize complementary DNA. Gene expression was measured using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green (Sigma, St. Louis, MO, USA). The Ct value, i.e., the number of cycles in which the fluorescence signal is statistically greater than that of the background, was used to quantify the expression of the target gene based on the 2-ΔΔCT method. The primer sets used are listed in Table 2. Expression of
PCR products generated from cDNA were cloned using TOPO vector (Invitrogen) to synthesize hybridization probes. Plasmids with the correct gene sequence verified were amplified using T7- and SP6-specific primers. The DIG RNA labeling kit (Roche, Indianapolis, IN, USA) was then used to transcribe DIG-labeled RNA probes. Testis tissues fixed in 4% paraformaldehyde were embedded in paraffin blocks and sectioned (5 μm thick) on slide glass. The sections were deparaffinized with xylene and rehydrated in DEPC-treated water containing a gradual alcohol series. Then, the sections were treated with 1% Triton X-100 for 20 min and washed twice with DEPC-treated phosphate buffered saline. The sections were then digested at 37oC with 5 μg/mL of proteinase K (Sigma) diluted in TE buffer. Next, the sections were exposed twice for 5 min in a buffer containing 0.25% (v/v) acetic anhydride and 0.1 M triethanolamine. The sections were exposed for 10 min at room temperature in a prehybridization mixture containing 50% formamide and 4X standard saline citrate (SSC). They were then incubated in a hybridization mixture and DIG-labeled cRNA probe for 16 h at 42oC. The sections were washed for 15 min in 2X SSC, 30 min in 1X SSC, 30 min in NTE buffer, and 30 min in 0.1X SSC. Normal sheep serum (2%) was used for blocking and sections were exposed for 16 h to sheep anti-DIG antibody conjugated with alkaline phosphatase. Signals for visualizing the expression of target genes were detected using solutions containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, and levamisole.
The statistical significance of quantitative PCR cells was confirmed based on the analysis of variance using SAS (SAS Institute). Differences with a probability value of
We first confirmed the mRNA level of the
Next, we identified the effects of high-cholesterol diets and quercetin supplementation on the expression of the
The expression of
In the testes of mice with a high-cholesterol diet,
Next, we examined the expression of genes involved in the uptake, transport, and synthesis of lipid content in the livers of mice fed diets containing cholesterol and quercetin. The expressions of carbohydrate-responsive element-binding protein (also known as
In this study, we identified changes in the expression of
Several studies have reported that the expression of proteins involved in lipid metabolism can affect testicular function in mice (Wang et al., 2017; Sharma et al., 2019). Lipids and cholesterol are essential for Sertoli cells to remodel the membranes of germ cells. Therefore, cholesterol is needed to produce large amounts of germ cells during spermatogenesis. The absence of the 24-dehydrocholesterol reductase gene, the enzyme responsible for synthesizing cholesterol, causes infertility in male mice (Wechsler et al., 2003). Cholesterol is not only a major component of cell membranes but is also involved in intracellular signaling. In addition, cholesterol can be converted into bioactive substances such as steroid hormones, vitamin D, and bile acids. The production of immature sperm is one of the main causes of male infertility. In a previous study, we identified changes in the expression of apolipoprotein D, a cholesterol-binding lipoprotein in male reproductive organs of rats fed a high-cholesterol diet (Lim et al., 2016). However, no studies, to the best of our knowledge, have identified the expression of genes involved in sperm maturation and fertilization in the testes of mammals fed cholesterol.
SPAM1 is the most widely conserved mammalian sperm antigen and is expressed in both the testes and sperm of humans and rodents (Martin-Deleon, 2011). SPAM1 plays an important role in the penetration of sperm into the cumulus during fertilization. The ADAM family of proteins are distributed throughout the male reproductive track, including in the testes and epididymis (Edwards et al., 2008). Adam1 and Adam2 are involved in membrane interactions of the sperm and oocytes. Sperm in male mice deficient in Adam1 and Adam2 lack mobility in the oviduct and ability to bind to the membrane of oocytes (Cho et al., 1998; Kim et al., 2006a). Adam5 is located on the surface of mature sperm in mice (Kim et al., 2006b). Adam29 is also involved in sperm-oocyte interaction, and reduced expression of Adam29 is considered a potential indicator of male infertility (Schiza et al., 2019). Therefore, SPAM1, ADAM5, and ADAM29 are considered molecular markers of sperm maturation and sperm fertilizing ability (Jaiswal et al., 2015; Liu et al., 2019). SRD5A1 and SRD5A2 belong to the steroid 5α-reductase family and are responsible for producing dihydrotestosterone, a derivative of testosterone (Saartok et al., 1984). In this study, a high-cholesterol diet reduced the expression of
Quercetin has cytoprotective effects by regulating lipid metabolism in many types of cells. In glial cells, expression of sterol regulatory element-binding protein (Srebp)-1, Srebp-2, and Mlxipl at the mRNA and protein levels was reduced by quercetin (Damiano et al., 2019). Quercetin-3-glucoside, a glucoside form of quercetin, can alleviate hyperlipidemia and hyperinsulinemia in mice fed a high-cholesterol diet (Mbikay et al., 2018). Moreover, quercetin contributes to alleviating male reproductive toxicity caused by environmental factors. Quercetin restores a reduced number of Sertoli cells in mice treated with diesel exhaust particles (Izawa et al., 2008). Quercetin also modulates proteins in apoptotic pathways such as Bax, Bcl-xl, and caspase-3 in germ cells of male mice and mitigates the testicular toxicity of 4-nitrophenol (Mi et al., 2013). Quercetin also mitigates increased cholesterol levels and decreased 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD activity in the testes of rats exposed to cadmium (Ujah et al., 2018). High-cholesterol diets cause oxidative stress, inflammatory responses, and mitochondrial dysfunction in the pancreas of rats; however, a diet supplemented with quercetin mitigates these detrimental effects (Carrasco-Pozo et al., 2016). In addition, our study found that quercetin can mitigate changes caused by a high-cholesterol diet in gene expression in the testes of mice. Quercetin also has a protective effect against the negative effects of the herbicide atrazine on serum testosterone levels and the expression of CYP17A1, a major protein responsible for steroidogenesis in the testes of rats (Abdel Aziz et al., 2018). In humans and rodents, testosterone is synthesized mainly in Leydig cells, and maintaining proper concentrations of testosterone is essential for spermatogenesis (Woolveridge et al., 1998). This study also revealed that finasteride, which inhibits the conversion of testosterone to its most active form, regulates changes in genes involved in spermatogenesis, similar to the effects of quercetin.
The liver is the main organ that regulates the homeostasis of cholesterol in the body. The liver regulates absorption and synthesis to prevent cholesterol from accumulating in the plasma or tissues (Goldstein et al., 2006). Mlxipl is a glucose-sensing transcription factor that has been reported to contribute to hepatoprotection by preventing excessive cholesterol synthesis (Zhang et al., 2017). Ppara is responsible for lipid metabolism in the liver and its transcriptional regulation has been shown to prevent hepatotoxicity from cholesterol overload (Lu et al., 2019). CD36 is involved in the intracellular uptake of cholesterol and fatty acids (Nassir et al., 2007). FABP1 combines with various hydrophobic ligands, including fatty acids in the liver, to help prevent cytotoxicity. A high-cholesterol diet is known to inhibit the expression of FABP1 (Newberry et al., 2003; Martin et al., 2006). FATP5 is also involved in the transport of fatty acids in the liver, and the deletion of FATP5 has been shown to lower serum cholesterol levels (Doege et al., 2006). CPT1 is responsible for fatty acid transport to the mitochondria, regulating fatty acid oxidation (Irvin et al., 2014). In this study, we found that high-cholesterol diets reduce the expression of lipid-related genes in the liver. The effects of quercetin in restoring the expression of genes that decreased under high-cholesterol conditions could be observed in
Taken together, we uncovered new genes related to sperm maturation and fertilization in male mice whose expression in the testes is altered with a high-cholesterol diet. In addition, confirmation that quercetin supplementation could reverse genetic changes caused by a high-cholesterol diet suggests that quercetin is a potential biologically active substance that can prevent male infertility caused by a high-cholesterol diet.
This research was supported by a grant of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) [grant number 2018R1C1B6009048]. We would also like to thank Institute of Animal Molecular Biotechnology in Korea University for supporting the publication fee.
C Yang, Ph.D. Candidate,
H Bae, Ph.D.,
G Song, Professor,
W Lim, Assistant Professor,