Icariin targets PDE5A to regulate viability, DNA synthesis and DNA damage of spermatogonial stem cells and improves reproductive capacity

Abstract

Icariin is a pure compound derived from Epimedium brevicornu Maxim, and it helps the regulation of male reproduction. Nevertheless, the role and underlying mechanisms of Icariin in mediating male germ cell development remain to be clarified. Here, we have demonstrated that Icariin promoted proliferation and DNA synthesis of mouse spermatogonial stem cells (SSCs). Furthermore, surface plasmon resonance iron (SPRi) and molecular docking (MOE) assays revealed that phosphodiesterase 5A (PDE5A) was an important target of Icariin in mouse SSCs. Mechanically, Icariin decreased the expression level of PDE5A. Interestingly, hydrogen peroxides (H₂O₂) enhanced the expression level of phosphorylation H2A.X (p-H2A.X), whereas Icariin diminished the expression level of p-H2A.X and DNA damage caused by H₂O₂ in mouse SSCs. Finally, our in vivo animal study indicated that Icariin protected male reproduction. Collectively, these results implicate that Icariin targets PDE5A to regulate mouse SSC viability and DNA damage and improves male reproductive capacity. This study thus sheds new insights into molecular mechanisms underlying the fate decisions of mammalian SSCs and offers a scientific basis for the clinical application of Icariin in male reproduction.

INTRODUCTION

Male infertility has been a serious issue for human reproduction worldwide, because around 50% of infertile couples are derived from the male factors.1 In North America, Europe, and Australia, the number of male spermatids has been decreased by 59.3% since the 1970s.2 Abnormal sperm parameters and male infertility can be derived from the imbalance of the reactive oxygen species (ROS).3 The treatment for male infertility mainly includes hormones, antioxidants, and traditional Chinese medicine. Notably, traditional Chinese medicine can improve the quality of spermatids.4 A major issue for treating male infertility using hormones is the safety concern. In addition, it remains to be defined about the efficacy of antioxidants to enhance semen qualities and DNA integrity of infertile men.5 Interestingly, traditional Chinese medicine has been employed to treat male infertility for numerous years because of its high safety and efficacy.

Icariin (C₃₃H₄₀O₁₅; molecular weight: 676.67 Da) is derived from Icariin herbal extract,6 and it assumes various kinds of pharmacological activities, including improvement of male reproductive function,7,8,9 antioxidative stress,10,11 anti-tumor,12 anti-aging,13 and anti-inflammation.14 Icariin can activate the NO-cyclic guanosine signaling pathway and improve male erectile dysfunction.15 Icariin has been shown to increase testosterone secretion and reduce ROS damage of mouse testes.16 Nevertheless, the mechanisms by which Icariin mediates mammalian spermatogenesis remained to be clarified.

Spermatogonial stem cells (SSCs) are the initial cells for retaining normal spermatogenesis and male fertility.17,18,19 The proper balance between the self-renewal and differentiation of SSCs is essential for maintaining normal male fertility. Once the fate decisions of SSCs become disrupted, mature spermatids cannot be produced.20 We have recently reported an interaction of opa interacting protein 5 (OIP5) with noncatalytic region of tyrosine kinase adaptor protein 2 (NCK2) to control the proliferation, DNA synthesis, and apoptosis of human SSCs.21 However, it remains largely elusive about the molecular mechanisms that regulate mammalian SSC fate determinations. Interestingly, traditional Chinese medicines have recently been shown to improve the male reproductive function of rats and regulate the proliferation of SSCs.22 Notably, SSCs can be utilized as an excellent stem cell model to explore the roles and mechanisms of drug candidates for treating male infertility. In the present study, we found that Icariin stimulated the proliferation and enhanced DNA synthesis of mouse SSCs. Significantly, we discovered that Icariin specifically targeted phosphodiesterase 5A (PDE5A) to increase the growth of mouse SSCs and reduce their DNA damage. Collectively, this study offers a novel molecular mechanism underlying mammalian spermatogenesis and it provides a new drug candidate for the treatment of male infertility.

MATERIALS AND METHODS

Reagent

Icariin was purchased from Zesheng Technology Co., Ltd. (Anqing, China), and it was dissolved with dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) for a stock solution.

Culture of mouse SSCs

Mouse C18-4 cell line was derived from primary mouse SSCs via transfecting with SV-40 large T gene, and these cells possess phenotypic characteristics of primary SSCs. Mouse SSCs were cultured with DMEM/F12 medium with the addition of 10% fetal bovine serum (FBS; Gibco, Grand Island, CA, USA), 2 mmol l⁻¹ glutamine, and 1% penicillin-streptomycin at 34°C in an incubator with 5% CO₂. The culture medium was changed every day, and mouse SSCs were passaged when cell confluency was reached 80%.

Mice

The C57BL/6 mice at 5–6 weeks old were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). These mice were housed in a specific pathogen-free (SPF) animal facility at School of Medicine, Hunan Normal University (HUNNU; Changsha, China). Animals used in this study were approved by the Biomedical Research Ethical Committee of HUNNU (Approval No. D2022046), and they were treated with sufficient food and water. The mice were classified into four groups (n = 8 in each group), including vehicle (the control mice), intraperitoneal injection of 100 mg kg⁻¹ body weight Icariin, intraperitoneal injection of 10 mmol kg⁻¹ body weight hydrogen peroxides (H₂O₂). The combination of Icariin and H₂O₂, and the treatments were performed every day for 3 weeks.

Methyl thiazolyl tetrazolium (MTT) assay

The MTT assay was used to assess the viability of mouse SSCs, and 6000 cells per well were plated in 96-well microplates (Thermo Fisher Scientific, Waltham, MA, USA). After culturing for 3 days, 2 mg ml⁻¹ tetrazolium (Sigma-Aldrich, Saint Louis, MO, USA) was added to cells and incubated for 5 h followed by adding 150 μl of DMSO to the cells. A microplate reader (Biotek, Winooski, VT, USA) was employed to measure the absorbance (optical density [OD] values) of cells at 490 nm.

The 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay

For EdU assay, 6000 cells per well were seeded into 96-well plates. The cells were treated Icariin and/or transfected with control small interfering RNA (siRNA [siCtrl]), PDE5A siRNAs (siPDE5A), with or without body weight H₂O₂. Subsequently, the cells were treated with 20 μmol l⁻¹ EdU (ApexBio, Houston, TX, USA) and incubated for 12 h, and 4% paraformaldehyde (PFA) was used to fix the cells. The cells were permeabilized by 0.5% Triton X-100 for 10 min. We conducted immunocytochemical staining to detect EdU-positive cells using an antibody against EdU, and we used Hoechst 33342 to label cell nuclei. Cell images were captured under a fluorescence microscope (DM3000; Leica, Wetzlar, Germany).

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)

We isolated total RNA from mouse SSCs using Trizol. The complementary DNAs (cDNAs) were generated by RT of messenger RNA (mRNA) using 0.6 μg of total RNA and 4 μl of 4× genomic DNA (gDNA) wiper mix by the RT kit (Vazyme, Nanjing, China). We performed the qPCR by a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) in terms of the manufacturer’s instructions.

Immunocytochemistry (ICC)

Mouse SSCs were incubated in 6-well plates that were precoated with coverslips, and they were treated with or without Icariin. We fixed the cells with 4% PFA for 15 min and washed them with PBS three times. Next, we permeabilized the cells using 0.1% Triton for 10 min and blocked them utilizing 5% bovine serum. Primary antibodies and secondary antibodies (Supplementary Table 1) were employed to incubate with the cells, respectively. The 4’,6-diamidino-2-phenylindole (DAPI) was used to label cell nuclei, and a fluorescence microscope (Leica) was applied to observe protein-expressing cells.

Supplementary Table 1.

Primary antibodies used in this study

Antibody	Catalog number	Company	Assay	Host
PDE5A	ab259945	Abcam (Cambridge, UK)	Western blot	Rabbit
p-PDE5A	GTX36930	GeneTex (Irvine, CA, USA)	Western blot	Rabbit
p-H2A.X	9718S	Cell Signaling Technology (Danvers, MA, USA)	Western blot, ICC	Rabbit
GAPDH	5174S	Cell Signaling Technology (Danvers, MA, USA)	Western blot	Rabbit
PCNA	sc-56	Santa Cruz (Santa Cruz, Bolivia)	Western blot	Rabbit
Histone-3	60932S	Cell Signaling Technology (Danvers, MA, USA)	Western blot	Rabbit

PDE5A: phosphodiesterase 5A; p-PDE5A: phosphorylation of PDE5A; p-H2A.X: phosphorylation of H2A.X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PCNA: proliferating cell nuclear antigen; ICC: immunocytochemistry

Transfection of siRNAs to mouse SSCs

We conducted the transfection of PDE5A siRNAs to mouse SSCs using Lipofectamine 6000 (Invitrogen, Carlsbad, CA, USA). In brief, 3 × 10⁵ cells per well were seeded into 6-well plates, and they were transfected with 50 nmol l⁻¹ PDE5A siRNAs or negative control siRNA. The targeting sequences of PDE5A were included as follows: siPDE5A-1: 5’-CGTGAACAACTCATACATA-3’; siPDE5A-2: 5’-GCCACTTAATATCCCAG-3’. Silencing efficiency of PDE5A was determined by RT-qPCR and Western blot.

Western blot

Mouse SSCs were plated at a density of 5 × 10⁵ cells per well, and they were incubated with DMEM/F12 containing 10% FBS. Radio immunoprecipitation assay (RIPA) buffer was employed to lyse cells, and centrifugation of cell lysates was performed at 16 060g (Heraeus Fresco17; Thermo Fisher Scientific) for 10 min at 4°C. Testis tissues were homogenized by Tissuelyser (Servicebio, Wuhan, China) in ice-cold RIPA. Protein concentrations of cells and testis tissues were measured by the BCA protein assay kit (Solarbio). Proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA). We blocked proteins using 5% non-fat milk in TBST at room temperature for 1 h and incubated them with the primary antibodies (Supplementary Table 1) overnight at 4°C and secondary antibodies for 1 h. ChemiDoc Touch (Bio-Rad) and Clarity™ Western ECL Substrate (Bio-Rad) were employed to detect proteins, and levels of proteins were quantified by Image J (NIH, Baltimore, MD, USA).

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

H&E was applied to stain testis sections. We fixed testicular tissues with 4% PFA for 24 h, and we embedded them in paraffin and sectioned them to 7 μm thickness.

For immunohistochemical staining of testicular sections, we deparaffinized and rehydrated them using xylene and 100%, 95%, and 75% ethanol. We blocked endogenous peroxidase activity using 3% H₂O₂. We incubated the sections with primary antibodies overnight at 4°C utilizing the goat hypersensitivity two-step detection kit (ZSGB-BIO, Beijing, China) according to the manufacturer’s instructions. DAB Substrate Kit (Cell Signaling, Beverly, MA, USA) was utilized to observe cell staining, and Gill’s hematoxylin (Solarbio) was employed to counterstain with cell nuclei. Immunostaining was determined by Image J after normalizing to the control.

Surface plasmon resonance iron (SPRi) affinity analysis

In order to seek the targets of icariin on mouse SSCs, affinity measurement was performed using SPRi. Briefly, we fixed mouse SSCs using photocross-linking SensorChip, and cell lysates were circulated on the surface chip of chip. SPRi affinity assay was employed to capture target proteins on the chip surface. The target proteins captured by proguanil were further identified by liquid chromatograph mass spectrometer (LC-MS), and we conducted data analyses using BetterWays (Guangzhou, China).

Sperm count

The seminiferous tubules of mice were removed and placed into petri dishes containing DEME/F12 medium. We incubated the seminiferous tubules at 34°C for 15 min to release sperm. We collected the sperm by centrifugating (Thermo Fisher Scientific) at 200g for 5 min, and we smeared sperm onto slides. Finally, we counted the cell number of sperm under a microscope (Leica).

Sperm staining

We smeared 10 μl of sperm-containing mixture onto cell slides, and we dyed it with 0.5% gentian purple alcohol solution for 10 min. We washed the cells with PBS, and a microscope (Leica) was employed to observe morphological abnormality.

Statistical analyses

We presented the data as mean ± standard deviation (s.d.) from at least three independent experiments. We determined the significant differences between two groups using the Student’s t-test and preformed multiple group comparisons using the one-way analysis of variance (ANOVA). Graphs were generated by GraphPad Prism 9.4 (GraphPad Software Inc., San Diego, CA, USA), while P < 0.05 was considered the statistical significance.

RESULTS

Icariin enhances mouse SSC viability and DNA synthesis

Our MTT revealed that Icariin enhanced cell viability of mouse SSCs at 2.5–15 µmol l⁻¹ and it decreased their growth at 20 µmol l⁻¹ (Figure 1a). Furthermore, proliferating cell nuclear antigen (PCNA) level (Figure 1b and 1c) and the percentages of EdU-positive cells (Figure 1d and 1e) were elevated by Icariin at 2.5–15 µmol l⁻¹ in mouse SSCs. Collectively, these data indicate that Icariin enhances cell viability and DNA synthesis of mouse SSCs.

Figure 1.

Icariin enhances the viability and DNA synthesis of mouse SSCs. (a) MTT assay was utilized to measure viability of C18-4 cells after 3 days of culture under different concentrations of Icariin. (b) Western blot was used to check the expression level of PCNA in C18-4 cells with treatment of Icariin at different concentrations for 24 h. (c) Statistical analysis of PCNA expression in b. (d) EdU incorporation assay showed the EdU-positive cells affected by Icariin at 2.5–15 µmol l⁻¹ in C18-4 cells. (e) Statistical analysis of EdU-positive cells in d. ^*P < 0.05 and ^**P < 0.01, the value of the indicated group compared with that in the control group (n = 3 for each group). MTT: methyl thiazolyl tetrazolium; SSC: spermatogonial stem cell; PCNA: proliferating cell nuclear antigen; EdU: 5-ethynyl-2’-deoxyuridine.

PDE5A has been identified as a target of Icariin in mouse SSCs

To uncover the mechanisms by which Icariin enhances the viability and DNA synthesis of mouse SSCs, we employed the SPRi target capture technique to identify the potential targets of Icariin. Notably, we found that Icariin treatment resulted in the strongest binding to PDE5A (Supplementary Figure 1a ^{(72.2KB, tif)} ). Meanwhile, the molecular binding energy of Icariin to PDE5A was shown to be −8.6 kJ mol⁻¹ using the molecular docking (MOE) techniques (Supplementary Figure 1b ^{(72.2KB, tif)} ). Together, these results suggest that PDE5A is a potential target of Icariin in mouse SSCs.

PDE5A silencing increases mouse SSC viability and DNA synthesis

To further explore the roles of PDE5A in mediating the fate decisions of mouse SSCs, we silenced PDE5A expression in C18-4 cells using siRNA technique. Our RT-qPCR and Western blot revealed that siPDE5A-1 and siPDE5A-2 decreased the expression level of PDE5A with a more silencing efficiency of siPDE5A-2 (Figure 2a–2c). In addition, PDE5A knockdown increased the viability of mouse SSCs as shown by MTT assay (Figure 2d). The EdU incorporation assay demonstrated that PDE5A silencing enhanced the percentages of EdU-positive cells (Figure 2e and 2f). Therefore, these data implicate that PDE5A silencing increases mouse SSC proliferation and DNA synthesis, which is consistent with the effect of Icariin on mouse SSCs.

Figure 2.

The effect of PDE5A silencing on the viability and DNA synthesis of C18-4 cells. (a) The qPCR assay was used to assess the impact of two PDE5A siRNAs on PDE5A mRNA of C18-4 cells. (b) Western blot demonstrated the influence of two PDE5A siRNAs on PDE5A protein of C18-4 cells. GAPDH served as a control of loading protein. (c) Statistical analysis of PDE5A protein expression in b. (d) The influence of PDE5A silencing on the viability of C18-4 cells. (e) EdU incorporation assay illustrated the impact of PDE5A siRNA-2 on DNA synthesis of C18-4 cells. (f) Statistical analysis of EdU assay of EdU-positive cells in e. ^*P<0.05 and ^**P < 0.01, the value of the indicated group compared with that in the control group. Ctrl: control; PDE5A: phosphodiesterase 5A; siRNA: small interfering RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EdU: 5-ethynyl-2’-deoxyuridine; mRNA: messenger RNA.

Icariin is an inhibitor of PDE5A

To further investigate whether PDE5A is an inhibitory target of Icariin in mouse SSCs, we utilized qPCR and Western blot to investigate the effect of PDE5A silencing on the Icariin-caused decrease of PDE5A expression. After PDE5A silencing, Icariin did not alter PDE5A expression at transcriptional or translational levels in mouse SSCs (Figure 3a–3c), indicating that the influence of Icariin on PDE5A depends on PDE5A. Meanwhile, our Western blot displayed that Icariin reduced the levels of PDE5A and phosphorylation of PDE5A (p-PDE5A) via a concentration-dependent manner (Figure 3d and 3e).

Figure 3.

The influence of Icariin and PDE5A silencing on PDE5A and p-PDE5A expression levels of C18-4 cells. (a) The qPCR was utilized to detect the effect of Icariin and PDE5A siRNA-2 on the level of PDE5A mRNA in C18-4 cells. (b) Western blot was employed to determine the influence of Icariin and PDE5A siRNA-2 on the level of PDE5A protein in C18-4 cells. (c) Statistical analysis of the expression of PDE5A protein in b. (d) Western blots showed the changes in the p-PDE5A and PDE5A expression levels of C18-4 cells treated with different concentrations of Icariin for 24 h. GAPDH was used as a loading control of proteins. (e) Statistical analysis of p-PDE5A and PDE5A protein expression in d. ^*P < 0.05 and ^**P < 0.01, the value of the indicated group compared with that in the control group (n = 3 for each group). Ctrl: control; PDE5A: phosphodiesterase 5A; p-PDE5A: phosphorylation of PDE5A; siRNA: small interfering RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; mRNA: messenger RNA; EdU: 5-ethynyl-2’-deoxyuridine; NS: no statistical difference.

H₂O₂ causes DNA damage of mouse SSCs

One of the main causes for male reproduction dysfunction is DNA damage.23 We thus determined whether H₂O₂ can cause DNA damage of mouse SSCs. We found that H₂O₂ reduced cell viability and DNA synthesis of mouse SSCs in a dose-dependent manner, as shown by MTT and EdU assays, respectively (Figure 4a–4c). Furthermore, ROS probe was employed to measure the ROS level of mouse SSCs, and H₂O₂ was seen to induce ROS production of these cells (Figure 4d and 4e). Additionally, H₂O₂ treatment led to the upregulation of phosphorylation of H2A.X (p-H2A.X), a marker for DNA damage, in these cells (Figure 4f and 4g), while H₂O₂ did not affect the PDE5A or p-PDE5A expression level in mouse SSCs (Figure 4h and 4i). Considered together, these data suggest that H₂O₂ causes DNA damage of mouse SSCs through the downstream of PDE5A.

Figure 4.

H₂O₂ inhibits viability and causes DNA damage in C18-4 cells. (a) MTT assay showed the effect of H₂O₂ at different concentrations on cell viability of C18-4 cells for 24 h. (b) EdU incorporation assay was utilized to detect EdU-positive cells in C18-4 cells affected by H₂O₂ at different concentrations. (c) Statistical analysis of EdU-positive cells in b. (d) The ROS kit was used to determine the changes in ROS levels of C18-4 cells after 2 h of treatment with H₂O₂ at different concentrations. (e) The ROS relative fluorescence intensity was calculated from the results of the five independent experiments in d. (f) Western blot showed p-H2A.X expression changes in C18-4 cells after 2 h of H₂O₂ treatment at different concentrations. (g) Statistical analysis of p-H2A.X protein expression in f._. (h) Western blot displayed the changes in expression levels of p-PDE5A and PDE5A in C18-4 cells after 2 h treatment of H₂O₂ at different concentrations. (i) Statistical analysis of p-PDE5A and PDE5A protein expression in h. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, the value of the indicated group compared with that in the control group (n = 3 for each group). NS: no statistical difference; MTT: methyl thiazolyl tetrazolium; PDE5A: phosphodiesterase 5A; p-PDE5A: phosphorylation of PDE5A; p-H2A.X: phosphorylation of H2A.X; H₂O₂: hydrogen peroxides; siRNA: small interfering RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; mRNA: messenger RNA; ROS: reactive oxygen species.

A to protect DNA damage of mouse SSCs caused by H₂O₂

Several assays were employed by us to determine whether Icariin protects DNA damage of mouse SSCs caused by H₂O₂. Our MTT assay displayed that Icariin reversed the H₂O₂-caused decrease in cell viability of mouse SSCs (Figure 5a). Our EdU incorporation assay (Figure 5b and 5c) and ROS probe analysis (Figure 5d and 5e) showed the impact of Icariin on DNA synthesis and ROS level of these cells. Western blot revealed that p-H2A.X level of mouse SSCs was up-regulated by H₂O₂ and decreased by Icariin, while the levels of PDE5A were unaffected by H₂O₂ alone in these cells (Figure 5f and 5g). To further demonstrate the antioxidant property, immunocytochemical staining illustrated that the fluorescence intensity of p-H2A.X in mouse SSCs treated with H₂O₂ alone was significantly stronger than that in the control, which was decreased by Icariin (Figure 5h). Taken together, these results mentioned above implicate that Icariin inhibits PDE5A expression and reduces the oxidative damage caused by H₂O₂ in mouse SSCs.

Figure 5.

Icariin protects DNA damage of C18-4 cells caused by H₂O₂. (a) MTT assay showed the effect of Icariin combination with H₂O₂ at different concentrations for 24 h on the viability of C18-4 cells. (b) EdU incorporation assay detected the EdU-positive cells in C18-4 cells with Icariin at different concentrations for 24 h and followed by H₂O₂ at 200 µmol l⁻¹ for 24 h. (c) Statistical analysis of EdU-positive cells in b. (d) Changes of ROS levels in C18-4 cells treated with Icariin for 24 h and followed by H₂O₂ at 200 µmol l⁻¹ for 2 h. (e) The ROS relative fluorescence intensity was calculated from the results of the five independent assays in d. (f) Western blot showed the changes of PDE5A and p-H2A.X protein expression in C18-4 cells treated with Icariin for 24 h and followed by H₂O₂ at 200 µmol l⁻¹ for 2 h. (g) Statistical analysis of PDE5A and p-H2A.X protein expression in f. (h) Immunocytochemical staining illustrated that the fluorescence intensity of p-H2A.X in C18-4 cells treated with Icariin for 24 h and followed by H₂O₂. ^*P < 0.05 and ^**P < 0.01, the value of the indicated group compared with that in the control group (n = 3 for each group). ^#P < 0.05 and ^##P < 0.01, vs H₂O₂ treatment (n = 3 for each group). NS: no statistical difference; MTT: methyl thiazolyl tetrazolium; PDE5A: phosphodiesterase 5A; p-PDE5A: phosphorylation of PDE5A; p-H2A.X: phosphorylation of H2A.X; H₂O₂: hydrogen peroxides; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ROS: reactive oxygen species; DAPI: 4’,6-diamidino-2-phenylindole.

Icariin improves reproductive functions of male mice in vivo

We finally evaluated the function of Icariin in regulating male reproduction in vivo. Male mice were classified into four groups, including the control mice, H₂O₂-treated mice, Icariin-treated mice, and H₂O₂ plus Icariin-treated mice. After 21 consecutive days of treatment, there was no obvious change in body weight, testicular weight, or morphological appearance among these groups of mice (Figure 6a–6c). Notably, we observed that the deformity ratios of sperm in the H₂O₂-treated mice were remarkably increased compared to the control mice, while Icariin treatment could reverse the H₂O₂-caused increase of the sperm deformity. No significant difference was observed in the abnormal morphology between the Icariin-treated mice and the control mice (Figure 6d). We illustrated representative sperm deformity after sperm staining (Figure 6e). For morphological examinations of testicular tissues, normal seminiferous tubules and all male germ cells were seen in Icariin-treated mice (Figure 6f). Significantly, the numbers of male germ cells including spermatids were reduced in the H₂O₂-treated mice compared to the control mice. In the H₂O₂ plus Icariin-treated mice, male germ cell numbers were increased (Figure 6f). Additionally, we found that p-H2A.X level was remarkably enhanced in the H₂O₂-treated mice compared to the control mice, whereas its expression level was significantly reduced in the H₂O₂ plus Icariin-treated mice. However, no significant difference in p-H2A.X level was seen between the Icariin-treated mice and the control mice. PDE5A expression level was decreased by Icariin, and H₂O₂ did not affect the expression of PDE5A (Figure 6g and 6h), which was consistent with our observations in vitro. Collectively, these results demonstrated that Icariin decreases the expression level of PDE5A, which reduces oxidative damage to the reproductive function of male mice caused by H₂O₂ in vivo.

Figure 6.

The influence of Icariin on reproductive reproduction of male mice in vivo. (a) Body and (b) testicular weight changes of male mice after being treated with H₂O₂, Icariin, and H₂O₂ plus Icariin for 21 consecutive days. (c) The representative testes of male mice after being treated with H₂O₂, Icariin, and H₂O₂ plus Icariin for 21 consecutive days (n = 8). (d) The abnormal rate of sperm of mice after being treated with H₂O₂, Icariin, and H₂O₂ plus Icariin for 21 consecutive days. (e) The appearance of sperm abnormalities in representative male mice after being treated with H₂O₂ for 21 consecutive days. (f) H&E staining of testicular seminiferous tubules of mice after being treated with H₂O₂, Icariin, and H₂O₂ plus Icariin for 21 consecutive days. (g) Western blot detected the expression changes of PDE5A and p-H2A.X proteins in the testis tissues of male mice after being treated with H₂O₂, Icariin, and H₂O₂ plus Icariin for 21 consecutive days. (h) Statistical analysis of PDE5A and p-H2A.X expression in g. ^*P < 0.05 and ^**P < 0.01, the value of the indicated group compared with that in the control group (n = 3 for each group). ^#P < 0.05 H&E, vs H₂O₂ treatment (n = 3 for each group). NS: no statistical difference; MTT: methyl thiazolyl tetrazolium; PDE5A: phosphodiesterase 5A; p-PDE5A: phosphorylation of PDE5A; p-H2A.X: phosphorylation of H2A.X; H₂O₂: hydrogen peroxides; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H&E: hematoxylin and eosin.

DISCUSSION

Icariin has the pharmacodynamic properties of regulating oxidative stress.10 However, the molecular mechanisms by which Icariin controls SSC fate determinations and antioxidant remain unknown. Here, we have demonstrated that Icariin inhibited PDE5A to enhance viability and DNA synthesis of mouse SSCs. Additionally, we found that Icariin reversed the DNA damage caused by H₂O₂ in mouse SSCs.

PDE5A belongs to the PDE5 superfamily, and it plays a key role in the decomposition of cGMP into GMP.24 PDE5A inhibitors, e.g., Sildenafil, Tadalafil, and Vardenafil, have been used as therapeutic agents for men with erectile dysfunction.25 The NO-cGMP pathway is activated through inhibiting PDE5A. Specifically, NO is released and cGMP can be accumulated in penile vascular smooth muscle cells, which leads to erection.26 Because of its universal expression in human tissues and unique pharmacological activity,27 PDE5A has attracted extensive attention in treating diseases, e.g., pulmonary hypertension,28,29,30 cardiovascular diseases,31,32,33 and diabetes.34,35,36 Using the SPRi technique, we found that PDE5A had the strongest binding to Icariin in mouse SSCs, and interestingly, Icariin could significantly reduce the expression level of PDE5A. Moreover, we found that PDE5A silencing enhanced DNA synthesis and viability of mouse SSCs. Therefore, Icariin might regulate the fate decisions of mouse SSCs by inhibiting PDE5A. This conclusion can be further verified by our findings that Icariin could not inhibit PDE5A after PDE5A silencing.

ROS imbalance has been reported to be one main pathogenesis for male infertility.3 It has been shown that H₂O₂ is a well-known reagent for establishing a DNA damage model.37,38 Our study demonstrated that H₂O₂ could induce oxidative damage in mouse SSCs. In addition, we observed that H₂O₂ did not affect the expression of PDE5A in mouse SSCs. Furthermore, we found that Icariin could reverse the H₂O₂-induced DNA damage in these cells. Significantly, our in vivo data revealed that Icariin did not affect body weight and testis size of mice. Our Western blot indicates that H₂O₂ resulted in DNA damage in the mouse reproductive system. Meanwhile, our immunohistochemical staining of the testicular tubules and the rate of sperm malformation reveal that H₂O₂ might have an effect on other cells in the testis, e.g., spermatocytes. We also explored the mechanism of Icariin in protecting the reproductive function of mice in vivo, and these data were consistent with our findings in vitro.

In summary, we have reported a vital role of Icariin in controlling DNA synthesis and viability of mouse SSCs and protects their DNA damage and male reproduction by targeting PDE5A in vitro and in vivo. As such, this study offers a novel mechanism that regulates the fate determinations of mammalian SSCs and it could provide a new traditional medicine candidate for treating male infertility.

AUTHOR CONTRIBUTIONS

TLL and CMH performed the experiments and wrote the manuscript. DX and ZRZ helped with the experiments. XPY and ZH were responsible for research design and revised the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.

Supplementary Figure 1

Identification of PDE5A as a target of Icariin in mouse SSCs. (a) SPRi detection with affinity protein comparison of Icariin after binding in C18-4 cells. (b) MOE was used to detect the binding of PDE5A to Icariin in C18-4 cells. SSC: spermatogonal stem cells; PDE5A: phosphodiesterase 5A; SPRi: surface plasmon resonance iron; MOE: molecular docking.