As men age, decreased testicular function and deterioration of sexual function, accompanied by mood changes, low libido, or physical transformations, commonly known as “andropause”, is observed [1
]. Typical physiological changes in aging men include sexual organ atrophy, delay in achieving full penile erection, poor quality of erection, and decline in orgasm intensity [2
]. Additionally, age-dependent changes are observed in the testes, both in the rate of steroidogenesis and spermatogenesis [4
Male reproductive function depends on the intermittent secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the pituitary gland under the influence of gonadotropin-releasing hormone [5
]. LH stimulates testicular Leydig cells to secrete testosterone, with a negative feedback mechanism to the hypothalamus to modulate LH secretion [6
]. FSH stimulates testicular Sertoli cells and promotes spermatogenesis.
Endocrine dysfunction in young males with pituitary deficiency, also known as Klinefelter’s syndrome, is primarily attributed to defects at the testicular region or defects in the regulation of hypothalamus/pituitary gland [7
]. However, changes in the regulation and progression of the hypothalamic–pituitary–gonadal axis, which is associated with decreased serum testosterone levels, might be one of the characteristic features of reproductive dysfunction in aging men [7
]. Furthermore, aging is also associated with increased free radicals and generation of other reactive species with decreased antioxidative defenses, resulting in chronic oxidative stress [8
Although aging is an inevitable process, delaying the progressive degenerative effects of aging, particularly in the testes, may help control male sexual dysfunctions (MSDs). Several nutritional and traditional herbs, such as Panax ginseng
, Withania somnifera
, Pausinystalia johimbe
, and Erycoma longifolia
, have been used for a considerable period of time in many parts of the world, including Asia, to treat MSD and shown potential in enhancing testicular functions disrupted by aging [10
]. These natural agents are attractive because they provide health benefits beyond those related to MSDs and are inexpensive compared to prescription medications. The basis of these nutrient herbal therapies used for MSD is that they help aging males improve their ability to overcome sexual dysfunction by increasing sexual stimulation as well as erectile, ejaculatory, orgasmic, and other responses [13
]. Most of these herbal nutrients have immense antioxidant, anti-inflammatory, and immunomodulatory properties, which helps regulate the altered oxidative defense system, spermatogenesis-related factors, and production of sex hormones.
Linn. (family, Cordycipitaceae; C. militaris
) is a valuable nutrient powerhouse and traditional medicinal mushroom that is well known since antiquity for its ability to revitalize various organ systems [15
]. As one of the earliest known natural remedy, C. militaris
is used extensively as a crude medicament and food in Asian countries [16
]. Pharmacologically, C. militaris
possesses antioxidant, anti-inflammatory, antiaging, anticancer, antiproliferative, antimetastatic, immunomodulatory, antimicrobial, antifibrotic, steroidogenic, hypoglycemic, hypolipidemic, antiangiogenetic, antidiabetic, neuroprotective, renoprotective, and pneumo-protective properties [17
]. Among the components of C. militaris
, cordycepin, also known as 3-deoxyadenosine, a purine nucleoside derivative, is a well-studied active constituent with significant biological properties, such as antitumor, antiviral, anti-inflammatory, and antiatherosclerotic effects [20
]. Several studies have demonstrated that cordycepin inhibits platelet aggregation [22
], induces apoptosis [23
], prevents hyperglycemia [24
], and inhibits the migration/proliferation of vascular smooth muscle cells and vascular neointimal formation [25
]. Cordycepin is also known to enhance sexual function in males [21
]. Structurally, cordycepin differs from adenosine by the absence of oxygen at the 3′ position of its ribose moiety. However, because of its structural similarity with adenosine, certain enzymes cannot discriminate between the two molecules. Therefore, cordycepin is readily phosphorylated intracellularly, enabling its participation in several physiological and biochemical reactions.
Previous studies have suggested that C. militaris
improves sexual function, supports the treatment of erectile dysfunction (ED), and acts as a pro-sexual agent. C. militaris
supplementation has also been found to improve sperm quality and quantity in rats [26
]. Previous reports from our laboratory showed that cordycepin attenuated age-related oxidative stress and ameliorated antioxidant capacity in old rats [27
]. However, the beneficial role of cordycepin and its modes of action in age-related testicular dysfunction remain unclear. In the present study, we investigated the ameliorative effects of cordycepin on age-related changes in sperm kinematics and expression of spermatogenesis-related key biomolecules, such as sex hormone receptors, oxidation-regulating enzymes, transcription factors as well as histone deacetylating sirtuin 1 (SIRT1) and autophagy-related mammalian target of rapamycin complex 1 (mTORC1) molecular changes in aged rats.
In the present study, the beneficial effects of COR and its mode of action on age-related sexual dysfunction was confirmed in experimental rat model. It is well known that age-associated alterations in testicular function are based on several parameters, including sperm motility, Sertoli cell number, sperm count in seminiferous tubules, and histopathological examination. Several studies have suggested that aging is associated with decreases in semen parameters, such as motility, sperm count, and Sertoli cell number [31
]. Aging is involved in the degeneration of germ cells and Sertoli cells, reduction in sperm production, immaturity of spermatozoa, and decrease in seminiferous tubular size. Furthermore, histomorphological examination of the tubular cross sections of aged rats confirmed deficient spermatogenesis [1
]. In agreement with the results of previous studies, our study showed reduced sperm motility, decreased seminiferous tubule size, and loss of sperm and Sertoli cell count per tubule in the AC group. However, COR treatment effectively ameliorated the age-related changes in histological parameters, such as the Johnsen’s score, seminiferous tubular size, and percentage of tubules containing sperms, sperm count, tubule and germ cell count. These changes were considerably improved by COR treatment.
To understand the cellular mechanism via which COR exhibits its beneficial effect on aging-induced testicular dysfunction, the protein and mRNA levels of spermatogenesis-related biomarker molecules were investigated. Reproductive function is regulated by sex steroids via their nuclear receptors in the hypothalamus and preoptic area. Studies have demonstrated a relationship between sex hormone receptors and aging-related testicular dysfunction [34
]. Sex hormone receptors, such as AR, LHR, and FSHR, play significant roles in maintaining male sexual function, and their altered expression may be involved in aging-related testicular disorders. Additionally, qualitative and quantitative changes in AR, the primary targets of androgenic steroids, as well as mutations in the gene encoding AR disrupt receptor sensitivity, leading to male reproductive system disorders [35
]. A reduction in the cytosolic AR population was detected in the hypothalamus of aged castrated rats [36
]. Furthermore, LHR and FSHR play crucial roles in the proper functioning of the hypothalamus–pituitary–testicular axis. LHR is necessary for testosterone production in Leydig cells, and cell-specific FSHR expression is involved in the response of Sertoli cells to FSH during spermatogenesis [37
]. In our study, we observed significant downregulation of AR, LHR, and FSHR in the testes of aged rats, which was significantly ameliorated by COR, suggesting that 20 mg/kg COR protects rats’ Leydig and Sertoli cells from aging-induced testicular dysfunction.
Key biomolecules such as inhibin-α, CREB-1, and nectin-2 are involved in testicular function and act as major spermatogenic factors [38
]. CREB-1, expressed during the mitotic phase of spermatocytogenesis and the differentiation phase of spermiogenesis, plays multiple roles in testicular development and function [38
]. CREB-1 may also regulate certain aging-related genes, such as the ataxia–telangiectasia mutated gene [42
]. Therefore, CREB-1 may be a key molecular regulator of testicular development and adult spermatogenesis [43
]. Nectin-2 is an important adhesion molecule present in the Sertoli–germ cell junction and is required for the maturation of spermatozoa in the seminiferous epithelium [44
]. Inhibin-α is an important biomarker of Sertoli cell activity in animals with impaired spermatogenesis, which negatively regulates FSH secretion from the anterior pituitary gland to maintain FSH homeostasis [45
]. In the present study, aged rats showed a significant reduction in the expression of inhibin-α, nectin-2, and CREB-1 at both the protein and mRNA levels compared with the YC rats, and this effect was significantly attenuated by treatment with 20 mg/kg COR (p
< 0.05). This indicates that aging significantly alters the expression of spermatogenesis-related molecules and that COR effectively restores these changes.
Previous reports have indicated that aging involves the accumulation of oxidative damage in cells and tissues and that oxidative stress is the main factor contributing to decreased organ and cell function during aging [8
]. High levels of polyunsaturated fatty acids accumulate in mammalian testes with aging, which are prone to free radical attack, creating an oxidative imbalance that results in impairment of steroidal testicular function [9
]. Previous reports from our group suggested that COR significantly attenuated age-related oxidative stress and decreased lipid peroxidation in aged rats [27
]. PRx4, an antioxidant enzyme that prevents oxidative stress-induced cell damage, is ubiquitously distributed in the mitochondrial matrix and expressed at high levels in reproductive organs [46
]. The levels of GST and its GSTm5 form, which occur in fibrous sheaths and promote the proliferation and differentiation of germ cells, may be altered during oxidative stress to combat attack by free radicals in the testes and spermatogenic cells [47
]. Similarly, GPx4, an antioxidant that protects against free radical-mediated damage to membrane lipids, proteins, and nucleic acids, has also been implicated as an important structural molecule in sperm maturation [49
]. Based on these reports, we evaluated the expression of key enzymes that regulate oxidation and play important roles in sperm function and spermatogenesis, such as GPx4, GSTm5, and PRx4. Consistent with the reported results, we observed that the levels of GPx4, GSTm5, and PRx4 in aged rats were downregulated than those in young control rats. COR (20 mg/kg) treatment of aged rats significantly ameliorated the reduction in protein and mRNA levels in the testis tissues (p
The role of SIRT1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function has been extensively studied [50
]. Sirtuins are associated with longevity and are involved in regulating male germ cell lifespan and aging-related decline in sperm quality [30
]. In contrast, the levels of mTORC1, a key modulator of autophagy- and age-related diseases, increases when food is abundant, prompting cells to increase their overall protein synthesis and proliferate. mTORC1 level decreases during unfavorable conditions, which reduces the overall production of proteins required for various biological activities, including events that are pivotal for spermatogenesis and the reproductive potential of males [51
]. Evidence suggests that mTORC1 regulates male fertility, and its inhibition is currently being investigated for the treatment of certain diseases, such as cancers and defects in male reproduction. Therefore, SIRT1 and mTORC1 can be used as biomarkers of aging and aging-related male reproductive conditions. We observed that the protein and mRNA levels of SIRT1 and p-mTORC1 in the AC group were significantly altered compared with those in the YC group (p
< 0.05) and that COR treatment (20 mg/kg) attenuated these changes in aged rats, suggesting that COR regulates and delays testicular aging. However, further studies are needed to clearly elucidate the detailed cellular mechanism via which COR improves testicular dysfunction associated with aging.