Hirsutella sinensis intensifies testicular function and spermatogenesis in male mice with high-fat diet-induced obesity

Abstract

Background:

Hirsutella sinensis (HS) is a mycelium isolated from the fruiting body of the medicinal mushroom Cordyceps sinensis. This study explored whether HS treatment affects reproductive dysfunction in a high-fat diet (HFD)-induced mouse model and regulates various mechanisms, focusing on oxidative stress, apoptosis, inflammation, and autophagy.

Methods:

Twenty-four C57BL/6J (B6) mice were randomly divided into a standard chow diet (NCD)- or HFD-fed group for 24 weeks. During the final 8 weeks, half of the HFD-fed mice were orally administered HS (HFD + HS). Biochemical markers, including glucose, insulin, triglycerides, and total cholesterol, were assessed, and hormones, including testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), were analyzed. Liver and testicular histology, as well as sperm quality markers such as sperm motility, sperm count, and percentage of sperm with normal morphology, were observed. The activities of the testicular antioxidants superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) and the products of lipid peroxidation, such as malondialdehyde (MDA), were measured. The protein expression levels of apoptosis-, autophagy- and inflammation-related markers were measured.

Results:

The HFD-fed mice had abnormal sex hormone levels, poor sperm quality, and a destroyed testicular structure, with increased oxidative stress and apoptosis in the testis. HS supplementation in HFD-fed mice attenuated testicular apoptosis by suppressing the Bax/Bcl-xl ratio and cleaved caspase 3 protein expression. The HS-treated mice exhibited improved reproductive function, possibly due to reduced oxidative stress and apoptosis, suggesting that HS has a protective effect against HFD-induced testicular damage.

Conclusion:

Male mice supplemented with HS exhibited attenuated poor semen quality and reduced testosterone levels brought about by HFD-induced obesity by reducing oxidative stress.

1. INTRODUCTION

Infertility is a reproductive disorder that affects nearly 8% to 12% of couples worldwide,¹ and male factors account for 40% to 50% of the primary and contributing causes of infertility.² Sperm abnormalities are common causes of male infertility, and Leaver³ reported that poor semen quality is responsible for up to 90% of infertility cases. Semen quality is an essential and critical indicator of male infertility, and semen quality not only represents a marker of sexual function but also has been found to be a reliable predictor of general health.^4,5 The results of a large, retrospective study involving approximately 5200 men showed that men with a lower sperm count were at greater risk of metabolic syndrome, osteopenia, and osteoporosis.⁶

Dietary factors such as hyper-caloric, hyper-saturated fat, hyper-trans fatty acids, and hypo-polyunsaturated fatty acid intake have been proven to have adverse effects on sperm function.^7,8 Moreover, an unhealthy diet consisting of high fat, high sugar, low vegetable, and low fruit consumption may increase leptin and cholesterol levels, as well as lipid-soluble toxins, thereby interfering with the hypothalamic-pituitary-gonadal axis, leading to hormonal imbalance and impaired spermatogenesis.⁹ Spermatogenesis is a complicated process of spermatozoa production from primary germ cells.¹⁰ It requires normal testicular function with proper hormonal stimulation, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone.¹¹

Oxidative stress is a critical factor in male infertility, causing apoptosis, DNA damage, protein oxidation, and lipid peroxidation during reproduction and leading to decreased fertility.^12,13 Inflammation with elevated pro-inflammatory cytokines indirectly increases reactive oxygen species (ROS) production and oxidative stress or directly influences testicular structure, testosterone biosynthesis, and spermatogenesis.^14,15 Recent studies revealed possible roles for autophagy in reproductive function,^16,17 and a mouse model of high-fat diet (HFD)-induced obesity showed that over-activation of autophagy impaired sperm production.¹⁸

Hirsutella sinensis (HS) is an anamorphic form of Cordyceps sinensis that has become an alternative to natural C. sinensis owing to the low production level and high cost of natural C. sinensis.¹⁹ Recently, HS has been found to exert protective effects against renal injury,²⁰ lung fibrosis,²¹ heart failure,²² and neurological defects²³ and to improve muscle function.²⁴ An in vivo experiment showed that HS modulated the intestinal microbial environment and exhibited an anti-obesogenic effect.²⁵ In addition, one of the active ingredients of HS, cordycepin (3’-deoxyadenosine), is known for its antioxidant and anti-inflammatory properties.²⁶ Little information is available concerning the effects of HS treatment on obesity-derived reproductive dysfunction; therefore, this study aimed to investigate the possible beneficial effects of HS treatment on testicular function, sperm quality, redox status, apoptosis, autophagy, and inflammation in mice fed an HFD.

2. METHODS

2.1. Animals, materials, and study design

HS (Strain:GKHS) was prepared and provided by Grape King Bio Ltd. (Taoyuan, Taiwan). The fungus was isolated from the fruiting body of C. sinensis, and the strain was authenticated by the Bioresource Collection and Research Center (Hsinchu, Taiwan), which was deposited as BCRC930209 in Taiwan. Briefly, mycelium colonies were primarily cultured in liquid culture media at 18°C, 120 rpm, and pH 5.0 for 14 days, followed by culture and fermentation. Mycelium powder was extracted with a 20-fold volume of 95% ethanol and concentrated by a rotary evaporator as the ethanol extract with a 20-fold volume of double-distilled water at 100°C for 30 minutes. The supernatant was collected by freeze-drying the water extract—the extracted product combined with the ethanol extract and water extract (1:1).

Male 8-week-old B6 mice purchased from the National Laboratory Animal Center (Taipei, Taiwan) were maintained in a well-controlled environment with a 12 h light/12 h dark cycle, a room temperature of 20°C to 26°C, and a relative humidity of 50% to 60%. After 2 weeks of adaptation, the mice were randomly divided into two groups, one receiving an NCD (n = 8; LabDiets 5010; supplying energy as 58% carbohydrate, 29% protein, 13% fat with total 3.02 kcal/g) and the other receiving an HFD (n = 16, Research Diets D12451; supplying energy from 35% carbohydrate, 20% protein, 45% fat with total 4.73 kcal/g). The HFD consisted of (g) sucrose (172.80), Lodex 10 (100.00), corn starch (72.80), fiber (50.00), casein (200.00), L-cystine (3.00), lard (177.50), soybean oil (25.00), minerals (50.00), vitamins (3.00), and dye (0.05), for a total of 858.15 g. After a 16-week diet induction period, half of the obese mice were orally fed 500 mg/kg H. sinensis for 8 weeks (HFD + HS, n = 8), while the mice in the NCD group and the remaining obese mice in the HFD group were orally fed an equal volume of solvent according to body weight.

2.2. Analyses of biochemical markers

Blood was collected quickly from the mouse hearts after fasting and deep anesthetization; it was then allowed to sit at room temperature for at least 30 minutes to form a clot, and serum was obtained by centrifugation at 1000 ×g for 10 minutes at 4°C. Serum levels of glucose, total cholesterol (T-CHOL), and triglyceride (TG) were measured at the National Applied Research Laboratories (Taipei, Taiwan). Serum insulin, FSH, LH, and testosterone levels were analyzed using commercial ELISA kits (Mercodia, 10-1247-01, Uppsala, Sweden; Cayman, No. 582701, Ann Arbor, MI; MyBioSource, MBS2507988, San Diego, CA; MyBioSource, MBS2514287) following the instructions provided by the manufacturers.

2.3. Analyses of sperm motility, sperm count, and sperm morphology

All semen analyses were performed by researchers who were blinded to the experimental groups. The sperm were flushed from the lumen of the vas deferens with diluted phosphate-buffered saline (PBS; Bioman, Taipei City, Taiwan), and the percentage of motile sperm was evaluated using an improved Neubauer counting chamber under a microscope (Leica, Wetzlar, Germany). After incubation at 37°C for 15 minutes, the sperm count was determined using a counter (Bio-Rad, Redmond, WA). Sperm morphology was assessed using an eosin Y staining assay, and the percentages of sperm with normal and abnormal morphologies were evaluated under a microscope.

2.4. Analyses of liver and testis histology

After fixation in 10% formalin, liver and testis samples were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). The stained slides were then imaged using a microscope and analyzed using ImageJ.

2.5. Analyses of apoptosis-, inflammation- and autophagy-related proteins in the testis

Testes were stored at −80°C after euthanasia. To prepare samples for western blotting, testis tissues were lysed in a mixture of RIPA buffer, protease inhibitor, and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA), and the total protein was quantified by DC protein assay (Bio-Rad). The extracted protein was loaded, separated by electrophoresis, and transferred onto polyvinylidene difluoride membranes. After being blocked in blocking buffer for 1 hour, the membranes were cut, incubated with the primary antibody overnight, and washed three times in TBST buffer. Then, the membranes were incubated with an anti-mouse (1:4000; sc-2005, Santa Cruz Biotechnology, Dallas, TX) or anti-rabbit secondary antibody (1:5000; sc-2005, Santa Cruz Biotechnology) for 1 hour and detected using chemiluminescence. The primary antibodies used were against caspase 9 (1:1000, #9508; Cell Signaling Technology, Danvers, MA), bax (1:1000, #2772; Cell Signaling Technology), bcl-xl (1:1000, ab32370; Abcam, Cambridge, MA), caspase 3 (1:750, #9662; Cell Signaling Technology), NF-кB (1:1000, E381; Abcam), TNF-α (1:1000, ab1793; Abcam), IL-6 (1:500, sc-57315), Beclin 1 (1:1000, sc-48341), p62 (1:1000, sc-48402), LC3B-II (1:1000, sc-271625) (all from Santa Cruz Biotechnology) and β-actin (1:10000, A5316; Sigma, Saint Louis, MO).

2.6. Analyses of redox status of the testis

Redox status, as demonstrated by the activities of antioxidants and the MDA content in the testis, was analyzed using assay kits purchased from Cayman (SOD, Item No. 706002; Catalase, Item No. 707002; GPx, Item No. 703102; TBARS, Item No. 10009055) following the protocols provided by the manufacturer.

2.7. Statistics

All the data are expressed as the means and SDs and were analyzed using the Kruskal-Wallis test, followed by the Dunn post hoc test with SAS Enterprise Guide 8.3 software. The results were considered significant at p < 0.05.

3. RESULTS

After the 24-week experimental period, the mice fed an HFD presented significantly elevated body weight, weight gain, and energy intake, with reduced food intake. Regarding the reproductive organs, the weights of the testis, epididymis, and vas deferens were not altered by the HFD. In addition, the epididymal fat mass increased approximately 2-fold in the groups fed an HFD (Fig. 1).

Fig. 1.

Effects of HS supplementation on body weight, weight gain, food intake, energy intake, and reproductive organs in HFD-fed mice. The values are presented as the means ± SDs. n = 8 per group; n = 6 per group (epididymal fat mass). *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

Compared with the NCD-fed mice, the HFD-fed mice showed notably abnormal blood metabolic parameters, manifested as increased glucose, insulin, and T-CHOL levels and decreased TG levels. However, the trends in these parameters in HFD-fed mice following HS treatment remained similar to those observed in HFD-fed mice (Fig. 2).

Fig. 2.

Effects of HS supplementation on serum glucose, insulin, total cholesterol, and triglyceride levels in HFD-fed mice. The values are presented as the means ± SDs. n = 6 per group. *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

The mean liver weight of the HFD group was markedly greater than that of the standard chow diet (NCD) group, and the HS group showed a decreasing trend with a more significant SD. In liver sections, there was steatosis and increased numbers and sizes of lipid vacuoles in hepatocytes in animals in the HFD group. The administration of HS improved hepatic morphology to some extent, as indicated by decreases in the numbers of small and large lipid droplets (Fig. 3).

Fig. 3.

Effects of HS supplementation on liver weight and liver histology in HFD-fed mice. The values are presented as the means ± SDs. n = 8 per group (liver weight); n = 4 per group (liver histology). *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

Although a similar sperm count was observed among the three groups, the other semen parameters, including sperm motility and normal sperm morphology, were significantly poorer in the HFD-fed mice than in those fed a regular diet. In contrast, the HS-treated HFD-fed mice exhibited enhanced sperm motility and improved normal sperm morphology, and there was no difference between the NCD group and the HFD + HS group. Mice fed an HFD also showed noticeable reductions in testosterone and LH levels compared with those of control mice, while the FSH level was the same among the three groups. Following HS treatment, decreased testosterone levels were restored, with an increasing trend in LH levels (Fig. 4).

Fig. 4.

Effects of HS supplementation on sperm quality and FSH, LH, and testosterone levels in HFD-fed mice. The values are presented as the means ± SDs. n = 8 per group (sperm quality); n = 5 per group (FSH, LH and testosterone). *p < 0.05. FSH = follicle-stimulating hormone; HFD = high-fat diet; HS = Hirsutella sinensis; LH = luteinizing hormone; NCD = standard chow diet.

No significant difference in the mean seminiferous tubule diameter (MSTD) was observed among the groups studied. Nevertheless, images of testis sections from the HFD group showed a disorganized and empty architecture with notably decreased spermatids in the lumen, with a consequent notably lower mean testicular biopsy score (MTBS, Johnsen score). Compared with HFD feeding alone, HFD feeding combined with HS treatment seemed to improve the impaired morphology of testicular seminiferous tubules and increased the MTBS, although the differences were not significant (Fig. 5).

Fig. 5.

Effects of HS supplementation on testicular histology and evaluations of MSTD and MTBS in HFD-fed mice. The values are presented as the means ± SDs. n = 5 per group. *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; MSTD = mean seminiferous tubule diameter; MTBS = mean testicular biopsy score; NCD = standard chow diet.

Regarding redox status in the testis, significantly lower activities of SOD, catalase (CAT), and GPx, in addition to a considerably higher MDA content, were present under HFD feeding. Treatment with HS notably reversed the increase in the MDA content to an average level. Moreover, the HFD + HS group exhibited trends toward elevated SOD, CAT, and GPx activities, although these elevations were not significant (Fig. 6).

Fig. 6.

Effects of HS supplementation on SOD, CAT, GPx, and MDA activity in the testes of HFD-fed mice. The values are presented as the means ± SDs. n = 4 per group (SOD, CAT, and GPx); n = 5 per group (MDA). *p < 0.05. CAT = catalase; GPx = glutathione peroxidase; HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet; SOD = superoxide dismutase.

HFD feeding increased the Bax/Bcl-xl ratio and cleaved caspase 3, whereas these effects were suppressed by HS treatment. Other markers involved in apoptosis, such as caspase 9, cleaved caspase 9, and caspase 3, were also examined, but neither the HFD nor the HS treatment had significant effects (Fig. 7 and Supplemental Fig. 1, http://links.lww.com/JCMA/A268).

Fig. 7.

Effects of HS supplementation on the protein expressions of apoptosis-related markers in the testes of HFD-fed mice. The values are presented as the means ± SDs. n = 3 per group. *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

Increased TNF-α protein expression but similar NF-кB and IL-6 protein expressions were observed in the HFD group compared to the NCD group. However, the increased expression of TNF-α remained in the HFD + HS group, indicating that HS treatment did not affect inflammation-related modulators (Fig. 8 and Supplemental Fig. 2, http://links.lww.com/JCMA/A268).

Fig. 8.

Effects of HS supplementation on the protein expressions of inflammation-related markers in the testes of HFD-fed mice. The values are presented as the means ± SDs. n = 3 per group. *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

The protein expression of the autophagy marker LC3B-II was significantly greater in HFD-fed mice than in NCD-fed mice. HS treatment had slight and nonsignificant reducing effects on LC3B-II. Compared to those in the NCD group, the protein expression levels of Beclin 1 and p62 in the HFD and HFD + HS groups were greater, but not significantly so (Fig. 9 and Supplemental Fig. 3, http://links.lww.com/JCMA/A268).

Fig. 9.

Effects of HS supplementation on the protein expressions of autophagy-related markers in the testes of HFD-fed mice. The values are presented as the means ± SDs. n = 3 per group. *p < 0.05. HFD = high-fat diet; HS = Hirsutella sinensis; NCD = standard chow diet.

4. DISCUSSION

As indicated by the results, obesity was successfully induced in C57BL/6J mice by an HFD, as demonstrated by dramatic increases in body weight, weight gain, energy intake, glucose, insulin, and cholesterol. On the other hand, the serum TG level was significantly lower than that of the control mice. Guo et al²⁷ reported a similar finding. They noted that rather than decreased amounts of dietary carbohydrates, increased amounts of dietary fat led to lower TG production and greater TG clearance, which may contribute to reducing TGs. Additionally, B6 mice have greater TG clearance due to strain characteristics.²⁸ Contrary to the expected results, HS treatment had no significant effect on weight gain, energy intake, epididymal fat accumulation, or metabolic profile in obese mice. There has been little discussion of the impact of H. sinensis on diet-induced obesity. Nevertheless, it has been reported that H. sinensis mycelium or polysaccharide fraction supplementation in HFD-fed mice prevents obesity by improving insulin resistance and gut barrier integrity and modulating the gut microbiota, lipid metabolism, and inflammation.²⁵ The differences between the results can be explained in part by the research methodology, including the duration and fat content of the HFD, as well as the dose and preparation of the treatment.

However, HS treatment improved hepatic steatosis, as characterized by reduced numbers and sizes of lipid droplets on microscopy images, and caused a decreasing trend in liver weight. These results were consistent with those of an earlier study by Wu, who reported that obesity-induced increases in lipogenesis and decreases in lipolysis and β-oxidation were attenuated by H. sinensis supplementation, which also reversed the progression of fatty liver.²⁵ This suggested that HS treatment does not affect weight loss or the metabolic profile. Nevertheless, the observation of noticeable pathological changes may have a positive impact on liver steatosis.

There is increasing evidence showing that HFD feeding causes a low testosterone level and poor sperm quality.^29,30 In this study, a reduced testosterone level, poorer sperm motility, and a lower percentage of sperm with normal morphology were observed after long-term HFD feeding. In addition, the HFD-fed mice had a lower epididymal weight, which plays a role in sperm maturation during the development of motility and affects the capability for fertilization³¹; this could partly explain the HFD-induced impaired sperm motility compared with the control. In addition, spermatogenic defects and a lower Johnsen score were observed in HFD-fed mice, as expected, because testosterone is known to be a vital regulator of spermatogenesis, which is required for maintenance of blood-testis barrier integrity, meiosis of spermatocytes, formation of adhering junctions between elongated spermatids and Sertoli cells, and spermiation.^32,33 Obesity-induced downregulation of gonadotropins can reduce testosterone levels,³⁴ which is consistent with the results of this study. In contrast, HS treatment increased the epididymal weight and testosterone levels, with an increasing trend in LH levels. It ameliorated the impaired spermatogenesis and sperm parameters affected by the HFD. Studies have indicated that C. sinensis has a structure similar to that of LH, which makes it capable of binding to LH receptors, and C. sinensis can activate the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways, thereby triggering testosterone production.^35,36 These results suggested an advantage of HS treatment in improving reproductive function.

The mechanisms involved in infertility, including redox status, apoptosis, inflammation, and autophagy in the testis, were analyzed in this study. Compared with the NCD-fed mice, the HFD-fed mice displayed decreased SOD, CAT, and GPx activities and increased MDA content, whereas HS supplementation led to reduced MDA content, indicating that the affected testicular antioxidant defense system was able to avoid peroxidative damage to a greater extent. Moreover, Chiu et al¹² reported that C. sinensis possesses anti-oxidative properties by promoting free radical scavenging activity and provided a perspective for confirming the anti-oxidative effects of HS treatment in further studies. High levels of ROS may influence apoptosis during spermatogenesis, activate caspases, induce apoptosis in testicular cells, and lead to testicular dysfunction. In the present study, HS supplementation in HFD-fed mice attenuated testicular apoptosis by suppressing the protein expressions of the Bax/Bcl-xl ratio and cleaved caspase 3. These results are partly supported by earlier published data showing that cordycepin, a bioactive ingredient of C. militaris, downregulated the protein expressions of cleaved caspase 3 and cleaved PARP, as detected by western blotting and immunohistology, in the testes of diabetic mice.³⁷ Further research focusing on HS treatment and the apoptotic pathway should be undertaken.

In terms of inflammation-related markers, both the vehicle-treated and HS-treated obese mice had a higher TNF-α protein expression but markedly lower NF-кB and IL-6 expressions than did the NCD-fed mice, revealing that HS treatment had no apparent effect on HFD-induced inflammation. However, Wu et al²⁵ reported lower serum and colonic IL-1β and TNF-α levels under H. sinensis supplementation. Huang et al²¹ used a bleomycin-induced pulmonary fibrosis mouse model. They reported that H. sinensis-derived diminished levels of ROS reduced the formation of the NRP3 inflammasome, inhibited the activation of caspase 1, and contributed to diminished inflammatory cytokines. Moreover, a previous study investigated the relationship between ROS and autophagy in testicular dysfunction and reported an adverse impact of autophagy on spermatogenesis.³⁸ Beclin 1 and LC3 are commonly used parameters of autophagy and are involved in the formation and maturation of autophagosomes. The HFD-fed mice had an increased protein expression of LC3B-II, indicating that HFD feeding induced autophagy. These results were in line with those of Latino et al³⁹ and Mu et al¹⁸; Mu et al also observed elevated protein expression of Beclin 1 in the semen of infertile obese men. In addition, an HFD-induced diabetic model demonstrated dual roles of autophagy in testis function; increased protein expression of Beclin 1 and the ratio of LC3-I/LC3-II in Sertoli cells and decreased expression of these proteins in Leydig cells were observed.⁴⁰ Further studies are required to provide insights into the role of autophagy in reproductive function. Despite the improvement in spermatogenesis, autophagy-related marker data from the mice that received HS treatment showed no improvement, and the present findings suggested that rather than inflammation and autophagy, HS supplementation in the present study had a protective effect against HFD-induced abnormal hormone levels and sperm dysfunction by ameliorating oxidative stress and apoptosis in the testis.

There were several limitations of this animal study: the lack of direct evidence of how HS suppresses testicular apoptosis, such as caspase activity assays and the identification and counting of testicular apoptotic cells via caspase immunostaining.⁴¹ The present study revealed that HS promoted alterations in testicular antioxidant status and reduced lipid peroxidation; the detection of ROS could further support the anti-oxidative attributes of HS. Obesity may have been shown to affect fertilization and pregnancy in both human⁴² and animal studies²⁹; however, studies have not performed mating trials, including studies on outcomes such as fertility rate, litter size, and the interval between litters, to provide more comprehensive estimates of the effects of HFD and HS treatment on male infertility. The HS ingredients that help improve spermatogenesis and testosterone levels by regulating redox status and apoptosis are also unclear. Future studies should be designed to clarify the above hypotheses and questions.

In conclusion, based on the results of this study, H. sinensis treatment could restore sperm production and inhibit oxidative imbalance and apoptosis to exert therapeutic effects against HFD-induced obesity-related testis damage and sperm dysfunction.

ACKNOWLEDGMENTS

Grape King Bio Ltd., Taoyuan County, Taiwan, funded this research.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data related to this article can be found at http://links.lww.com/JCMA/A268.