Regulation of testosterone synthesis by circadian clock genes and its research progress in male diseases

Abstract

The circadian clock is an important internal time regulatory system for a range of physiological and behavioral rhythms within living organisms. Testosterone, as one of the most critical sex hormones, is essential for the development of the reproductive system, maintenance of reproductive function, and the overall health of males. The secretion of testosterone in mammals is characterized by distinct circadian rhythms and is closely associated with the regulation of circadian clock genes. Here we review the central and peripheral regulatory mechanisms underlying the influence of circadian clock genes upon testosterone synthesis. We also examined the specific effects of these genes on the occurrence, development, and treatment of common male diseases, including late-onset hypogonadism, erectile dysfunction, male infertility, and prostate cancer.

INTRODUCTION

The circadian clock system has been shown to regulate a variety of biological processes in living organisms, such as hormone secretion, body temperature, sleep, and cell cycle progression.1,2,3 Circadian clock genes, including core and peripheral clock genes, refer to a group of genes that are involved in the regulation of circadian rhythms.4 Testosterone is an important hormone synthesized by Leydig cells and has crucial roles in the development and maintenance of male reproductive function.5 Earlier studies have revealed that the synthesis of testosterone in males is closely related to circadian clock genes. Disruption of circadian rhythms can lead to dysregulation of clock genes, and mutations or deletions in individual clock genes can result in abnormal testosterone levels and affect the reproductive health and overall health of men.6,7,8 It was reported that factors such as shift work, night-time light exposure, and social jet lag can all lead to disruptions in circadian rhythms, thereby affecting testosterone synthesis.9 The occurrence of diseases of the male reproductive system, such as prostate cancer (PCa), has also been associated with disrupted circadian rhythms.10 Therefore, clarifying the relationship between circadian rhythms and testosterone synthesis may provide new perspectives for the treatment of male diseases in clinical practice. For instance, simulating the circadian variation of endogenous testosterone through timely drug administration was shown to provide a therapeutic effect on late-onset hypogonadism (LOH) in men.11 Additionally, adjusting lifestyles, improving sleep quality, and optimizing lighting conditions can help restore normal circadian rhythmicity, which may potentially improve testosterone levels and related health problems.12 Overall, in-depth research on the correlation between circadian rhythms and testosterone synthesis is of important clinical significance to the future management and treatment of male diseases.

REGULATORY MECHANISMS OF THE CIRCADIAN CLOCK SYSTEM

The central control site of circadian clock rhythm is the suprachiasmatic nucleus (SCN) of the hypothalamus,13 which directly receives light information through the retinohypothalamic tract to guide the body’s circadian clock to enter a 24-h cycle rhythm. Then, the SCN transmits the phase information to other regions of the brain, as well as cells of peripheral tissues and organs, through a combination of neural, humoral, and systemic signals to drive the peripheral biorhythms.14 Earlier research has shown that circadian clock genes are expressed in the SCN of the hypothalamus, and also widely expressed in a range of peripheral tissues and organs, including the testis, and that the joint regulation of the central and peripheral circadian clocks constitutes a complete clock system.15 At the molecular level, circadian clock oscillations are formed by the interaction between positive and negative feedback transcription and translation loops composed of core genes and their encoded proteins. More specifically, the core genes include circadian locomotor output cycle kaput (CLOCK), brain and muscle arnt-like protein 1 (BMAL1), Per1/2/3 of the period gene family, Cry1/2 of the cryptochrome family, nuclear receptor subfamily 1 group D member 1/2 (NR1D1/2), and retinoic acid receptor-related orphan receptor α/β (RORα/β).2 BMAL1 and CLOCK can bind with each other through the PER-ARNT-SIM (PAS) domain to form the CLOCK:BMAL1 heterodimer, which initiates the transcription of PER and CRY mRNA by recognizing and binding to the E-box in the downstream gene promoters. The translated PER and CRY proteins can then bind with each other to form heterodimers, thereby inhibiting the transcriptional activity of CLOCK:BMAL1 and forming a transcription–translation negative feedback loop.16 Upon degradation of the PER and CRY proteins, the transcriptional activity of CLOCK:BMAL1 will be restored, so that the next round of gene transcription can be initiated (one such cycle is approximately 24 h).16 Additionally, the CLOCK:BMAL1 heterodimer can also initiate the transcription of a second feedback loop gene, which involves two types of transcription factors, namely NR1D1/2 and RORα/β, in the circadian clock system. Initially, CLOCK:BMAL1 promotes the transcription of REV-ERBα/β (NR1D1/2) and RORα/β, and the accumulated NR1D1/2 and RORα/β proteins interact with the BMAL1 promoter in the nucleus to compete for binding sites exhibiting retinoic acid-related orphan receptor response elements (RORE).17 Specifically, the ROR protein initiates the transcription of BMAL1, while the REV-ERBα protein inhibits its transcription, and the two coordinate with aforementioned loops to maintain the overall stability of the circadian clock system.17 In addition to transcriptional activation and inhibition, post-translational modifications (e.g., phosphorylation, ubiquitination, and acetylation mediated by different intracellular signaling pathways) also play important roles in maintaining the stability of biorhythms. For example, casein kinase 1, a key kinase involved in the phosphorylation of PER and CRY, can promote the degradation of PER via the phosphorylation process. The stability of BMAL1 is affected by ubiquitination, and the acetylation of BMAL1 has been shown to promote its binding to CRY.1 Moreover, it was revealed that CLOCK:BMAL1 initiates the transcription of downstream clock-controlled genes by activating the E-box to regulate the rhythmic changes in various physiological functions in mammals, as shown in Figure 1.18,19

Figure 1.

The circadian clock mechanism. CLOCK: circadian locomotor output cycle kaput; BMAL1: brain and muscle arnt-like protein 1; PER: period gene family; CRY: cryptochrome; NR1D1/2: nuclear receptor subfamily 1 group D member 1/2; RORα/β: retinoic acid receptor-related orphan receptor α/β; CCG: clock-controlled genes; CK1: casein kinase 1. Created by Figdraw.com.

EXPRESSION OF CIRCADIAN CLOCK GENES IN TESTICULAR TISSUES

Circadian clock genes are expressed in the SCN of the central nervous system and in various peripheral organ tissues and cells, including the testis.11 Per1 was the first biological clock gene discovered to be expressed in testicular tissue, followed by Per2, Per3, Cry1, Cry2, BMAL1, and CLOCK.20 However, there has been controversy over whether the expression of circadian clock genes in the testis manifests circadian rhythmicity. An earlier study reported that the expression of certain circadian clock genes (Bmal1, Nr1d1, Dbp, Per1, and Per2) was subjected to minor circadian rhythmic changes in the testes of mice.21 Meyer and Lerchl22 detected circadian rhythmicity in the expression of Bmal1, Per1, Per2, Clock, Cry1, and Cry2 in the testes of different strains of hamsters. Another group of researchers also detected circadian expression of Per1 in hamster testes.23 In contrast, Morse et al.24 found that Per1 and Bmal1 were consistently expressed in the testis 24 h throughout a day and lacked circadian rhythmicity. Moreover, the results of real-time PCR also indicated that the mRNA levels of some clock genes (Per1, Per2, Cry1, Cry2, Clock, Bmal1, and Nr1d1) exhibited no circadian rhythmic fluctuations in the testes of mice.25,26 Notably, in all the above studies, the expression of circadian clock genes was measured at several time points within approximately 24 h for the purpose of testing the presence of rhythmic oscillations. In contrast, Nishide et al.27 observed the real-time expression rhythm of Per2 in testicular tissue slices from transgenic rats by employing bioluminescence technology and detected significant circadian rhythmicity in the testicular expression of Per2 in both 5-day-old and adult rats. Although circadian clock genes have been confirmed to be expressed in the testis, current research findings suggest that different clock genes exhibit distinct testicular expression patterns because of species variations, the complexity of cell types, and the lack of cell type differentiation in most studies (the amplitude and phase of gene expression oscillations may vary among different types of cells in the testis).

Leydig cells

The testis is composed of Leydig cells, Sertoli cells, and spermatogenic cells at different developmental stages.28 Leydig cells are mainly responsible for the synthesis of androgens, which can promote spermatogenesis in the form of paracrine actions, and also regulate a range of endocrine processes outside the testis.29 The widespread expression of circadian clock genes in various testicular cells suggests that these clock genes may play crucial roles in regulating reproductive function in mammals. Although controversy remains over whether circadian clock genes are rhythmically expressed in testicular tissues, their expression in Leydig cells has been demonstrated to exhibit significant circadian rhythmicity. For instance, Bmal1, Per1, Per2, Per3, Cry1, Cry2, and Nr1d1 were all found to be expressed with circadian rhythmicity in the Leydig cells of rats and mice.8,30,31,32,33,34,35,36

Spermatogenic cells

Spermatogenic cells mainly consist of spermatogonia, spermatocytes, and spermatids. Spermatogonia, the source of spermatogenesis, can undergo self-renewal through mitosis and differentiate into spermatocytes, which then form haploid spermatids through meiosis.37 Haploid spermatids undergo circular and elongated cell stages and ultimately differentiate into morphologically mature sperm.37 Circadian clock genes have been shown to be expressed in multiple cell types during spermatogenesis. For instance, Bmal1 and Clock are expressed in spermatogonia, spermatocytes, and round sperm cells.7,24,38,39,40 Cry1 is expressed in spermatogonia and spermatocytes.41,42 Per1 is expressed in spermatogonia, spermatocytes, and elongated sperm cells,24,25,40 and Nr1d1 is only expressed in spermatogonia.6,43 Although circadian clock genes are widely expressed at various levels in spermatogenic cells, the expression of Per1 and Clock in spermatogenic cells does not exhibit circadian rhythmicity.44 Overall, circadian clock genes may exert important effects on the process of spermatogenesis through pathways including retinoic acid signaling, homologous recombination, and chromosomal mimicry.45

THE CIRCADIAN CLOCK SYSTEM REGULATES THE RHYTHMICITY OF TESTOSTERONE SECRETION

The secretion of testosterone manifests as different rhythms, including entire lifecycle, annual, and daily rhythms.46,47 As men age, their serum testosterone levels exhibit a gradually declining trend.48 The circadian rhythm of serum testosterone levels in healthy elderly men is weakened compared with that in healthy young men.49,50 It was reported that Leydig cells in aged rats produced less cyclic adenosine monophosphate (cAMP) and testosterone under the action of luteinizing hormone (LH) as compared with cells in younger rats.51 Consistent with animal studies, the level of testosterone production stimulated by the administration of human chorionic gonadotropin in elderly men was lower than that in young men,52 indicating a reduced responsiveness of Leydig cells to LH as men age. Although serum LH levels seem not to change significantly with age, the frequency and amplitude of LH pulses are correlated with age. Research showed that healthy young men exhibited circadian changes in LH pulses, with a decrease in nighttime frequency and an increase in amplitude.53 In contrast, healthy elderly men seemed to have lost circadian rhythmicity in LH pulses.53 Although their pulse frequency was similar to that of young men, the amplitude was reduced, leading to cyclical changes in the rhythm of testosterone secretion.53 Testosterone levels in bulls were found to exhibit seasonal and diurnal rhythms,54 while serum testosterone levels in goats, rats, mice, and young men were found to exhibit significant circadian rhythms.8,55,56,57 Overall, the secretion of testosterone manifests multiple patterns of rhythmicity, and the underlying mechanisms may be related to rhythmic regulation of the secretion of various reproductive hormones in mammals, such as LH and testosterone, by the circadian clock system.58,59,60

MECHANISMS OF CIRCADIAN CLOCK GENES IN REGULATING THE RHYTHMICITY OF TESTOSTERONE SYNTHESIS AND SECRETION

Circadian clock genes regulate the rhythmicity of testosterone synthesis and secretion through multiple modes, mainly via the central regulation of SCN, as well as the peripheral regulation of testosterone synthase, oxidative stress, and autophagy in Leydig cells (Figure 2).

Figure 2.

The effects of circadian clock genes on testosterone synthesis. CLOCK: circadian locomotor output cycle kaput; LHR: luteinizing hormone receptor; LH: luteinizing hormone; SCN: suprachiasmatic nucleus; HDL: high-density lipoprotein; SR-BI: scavenger receptor Class B; MT1: melatonin receptor 1; ROS: reactive oxygen species; CRH: corticotropin-releasing hormone; CYP11A1: cytochrome P450 cholesterol side chain lyase; HSD-3β: hydroxysteroid dehydrogenase-3β; HSD-17β: hydroxysteroid dehydrogenase-17β; BMAL1: brain and muscle arnt-like protein 1; PER1/2: period 1/2; NR1D1: nuclear receptor subfamily 1 group D member 1/2; StAR: steroidogenic acute regulatory protein; cAMP: cyclic adenosine monophosphate; GnRH: gonadotropin-releasing hormone. Created by Figdraw.com.

Central regulation of SCN

The SCN perceives ambient light through the retinal hypothalamic pathway, then converts photoperiodic signals into endocrine signals and transmits the signals to Leydig cells through various hormones, including melatonin and LH.33,36 In mammals, most circulating melatonin originates from the pineal gland,61 where the SCN plays a regulatory role in melatonin secretion.62 Once synthesized by the pineal gland, melatonin has high diffusivity and will be rapidly released into the cerebrospinal fluid and blood. Melatonin levels in body fluids and blood exhibit clear circadian rhythms under SCN regulation, with low secretion levels during the day time and peak levels at night.63 More specifically, melatonin regulates the secretion of testosterone by acting on the related factors as follows. (1) Melatonin inhibits the release of gonadotropin-releasing hormone (GnRH) in the hypothalamus by enhancing the activity of γ-aminobutyric acid receptors.64 (2) GnRH stimulates the release of LH from the pituitary gland by increasing the concentration of intracellular free calcium ions, and there are approximately synchronous diurnal fluctuations in LH and testosterone levels in healthy adult men.65 The rhythmic secretion of LH activates the expression of a series of enzymes and proteins involved in steroidogenesis through the LH receptor-cAMP signaling pathway,66,67 thereby promoting testosterone synthesis.68 (3) In addition to inhibiting testosterone synthesis induced by LH and GnRH, melatonin can also regulate testosterone production by binding to the melatonin receptor 1 expressed on Leydig cells to act on the corticotropin-releasing hormone (CRH).69 Secreted by Leydig cells, CRH is an important negative feedback regulator of testosterone production induced by gonadotropins.70 Melatonin can significantly increase the expression level of CRH mRNA in Leydig cells.69 CRH then inhibits the expression of steroidogenic acute regulatory protein (StAR) through the cAMP-dependent signaling pathway.71,72 It was reported that the circadian rhythm of testosterone secretion was markedly affected in melatonin receptor 1 gene knockout mice.20 The expression of StAR and testosterone synthesis were both increased in Leydig cells treated with CRH antagonists, indicating that melatonin indirectly exerts its regulatory effects on testosterone secretion through the CRH system,72 forming a circadian rhythm.61,73,74 And (4) elevated serum testosterone levels inhibit the production of GnRH and LH, thereby forming negative feedback.75

Peripheral regulation

The peripheral circadian clock genes in Leydig cells have a significant impact on testosterone synthesis.8,33,36 Genomic research has shown that the expression levels of approximately one-third of genes in cells change with the circadian rhythm, with most of these genes directly regulated by Bmal1 and Clock.76 More specifically, Bmal1 and Clock regulate testosterone levels by directly affecting the expression of genes related to testosterone synthesis. StAR, cytochrome P450 cholesterol side chain lyase (P450scc, also known as Cyp11a1), hydroxysteroid dehydrogenase-3β (HSD-3β), and hydroxysteroid dehydrogenase-17β (HSD-17β) are important rate-limiting enzymes in the testosterone synthesis pathway in Leydig cells.77 StAR is responsible for mediating the transport of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane.52,78 CYP11A1 acts as a catalyst for the conversion of cholesterol on the inner surface of the mitochondrial matrix membrane into pregnenolone.52,78 HSD-3β is involved in the enzymatic hydrolysis of pregnenolone to progesterone, which is ultimately converted to testosterone under the action of HSD-17β. In the Leydig cells of mice, the cis-regulatory elements controlled by circadian clock genes, including E-box, RORE, and D-box motifs, were discovered in the Star and Cyp11a1 promoters.30 Additionally, analysis of the Star and Hsd-17β promoters in the Leydig cells of rats and goats demonstrated that RORE motifs were also present upstream of their transcription start sites.8,79 BMAL1 and CLOCK directly regulate the expression of Star by binding to the E-box on the Star promoter.7 Changes in the expression levels of BMAL1 and CLOCK can directly affect the expression of genes related to testosterone synthesis. For instance, it was found that overexpression of Bmal1 in the Leydig cells of goats significantly increased the mRNA and protein levels of Star and Hsd-17β.8 In Bmal1 knockout (Bmal1^−/−) infertile mice, testosterone levels and the expression levels of Star and Cyp11a1 in Leydig cells were reduced.7,80 Also, Bmal1 knockdown in the mouse Leydig cell (TM3 cell) line was found to lower the expression of Star, Cyp11a1, and Hsd-3β.35

Downstream genes of Bmal1 and Clock, Per1/Per2 and Nr1d1, also have significant effects on testosterone synthesis. The expression levels of Cyp11a1 and Hsd3b, as well as the serum free testosterone levels, were downregulated in testes of Per1/Per2 double-knockout male mice compared with wildtype male mice.81 Nr1d1 can directly regulate the expression of genes related to testosterone synthesis by binding to the RORE in their promoters,6 and can also indirectly regulate these genes through the feedback inhibition of Bmal1 transcription by binding to the RORE in the Bmal1 promoter.17 After treatment of primary rat Leydig cells with a Nr1d1 agonist (GSK4112), the mRNA expression levels of Bmal1, StAR, Cyp11a1, and Cyp17a1 were decreased, while treatment with a Nr1d1 antagonist (SR8278) resulted in the opposite effect.82 The Nr1d1 agonist (SR9009) significantly shortened the Bmal1-Luc oscillation period and reduced its amplitude in TM3 cells.31 However, not all circadian clock genes have direct effects on testosterone synthesis. For instance, serum and testicular testosterone levels in mice were not affected after the disruption of Cry1.41

Furthermore, peripheral circadian clock genes also influence the oxidative stress in Leydig cells. Tntracellular redox state changes with the circadian rhythm. As one of the products of redox, reactive oxygen species (ROS) are produced and accumulated in large quantities in cells and can induce DNA damage, impair protein function, and induce lipid peroxidation. Leydig cells in mammals are rich in polyunsaturated fatty acids, which are easily attacked by ROS, leading to oxidative stress.83,84 ROS can inhibit testosterone synthesis by reducing the expression of StAR.85,86,87,88 Conversely, ROS can also affect LH secretion from the pituitary gland by promoting cortisol secretion, thereby indirectly reducing testosterone production in Leydig cells.85 Overall, Bmal1, Per2, and melatonin can inhibit oxidative stress by reducing ROS formation,89,90,91,92 thereby promoting testosterone synthesis in Leydig cells.

Additionally, the impact extends to autophagy processes within Leydig cells. Autophagy refers to an intracellular degradation process that targets the cytoplasmic components of lysosomes for degradation, to maintain cellular homeostasis and provide substrates for synthesis reactions.93 It has been demonstrated that changes in the number of intracellular autophagosomes exhibit a diurnal rhythm.94 Specifically, the volume and density of autophagosomes are highest in the afternoon and gradually decrease during the night time,95 indicating a possible connection between autophagy and the circadian clock system. The expression levels of autophagy genes and the number of autophagosomes were significantly reduced in Bmal1^−/− mice.96 It was also reported that decreased Per2 expression resulted in a reduced autophagy flux,97 and that Nr1d1 regulated autophagy by inhibiting the expression of the autophagy gene autophagy-related gene 5 (ATG5).98 From the above findings, it may be speculated that the circadian clock regulatory network exerts direct effects on the expression of autophagy genes and the formation of autophagosomes. In the testes of rats, Leydig cells exhibited higher levels of autophagy than Sertoli cells.99 Moreover, the autophagy activity level has been associated with the steroid release level.100 Autophagosomes selectively target organelles that play important roles in steroid synthesis, such as mitochondria and smooth endoplasmic reticulum in Leydig cells. In the case of reduced autophagy, the removal of dysfunctional mitochondria in Leydig cells tends to decrease, leading to the accumulation of ROS that ultimately affects testosterone synthesis.101 Free cholesterol is a precursor for testosterone biosynthesis. Cholesterol uptake by Leydig cells depends on the scavenger receptor Class B, type I, which is conducive to selectively grabbing the cholesterol derived from lipoproteins.102 Autophagy promotes the uptake of cholesterol into Leydig cells by eliminating the Na/H exchange regulatory factor 2, which is a negative regulatory factor of the scavenger receptor.103 In summary, circadian clock genes regulate testosterone synthesis in an indirect manner by affecting the autophagy level in Leydig cells.

RESEARCH PROGRESS ON THE CIRCADIAN CLOCK SYSTEM IN THE OCCURRENCE, DEVELOPMENT, AND TREATMENT OF MALE DISEASES

Circadian rhythm disorders can lead to a variety of pathological conditions.104 Rotating shift work (RSW) and insufficient sleep at night are both important factors causing circadian disturbances. The International Labor Organization and the European Foundation for the Improvement of Living and Working Conditions compared the shift work situation of approximately 1.2 billion workers in 187 countries/regions and concluded that 10%–30% of workers had at least one night shift per month.105 Clinical studies revealed that RSW would lead to a decrease in the total testosterone and pregnenolone levels in men106,107 and a significant delay in the peak time of serum testosterone.108 A meta-analysis involving 18 clinical studies and 252 males showed that complete nighttime sleep deprivation could lead to a decrease in serum testosterone levels in men,109 possibly due to alterations in the epigenetic and transcriptional profiles of core circadian clock genes in key metabolic tissues.110 In adult rat models simulating the disruption of human circadian rhythmicity by RSW, researchers also observed a significant decrease in serum testosterone levels.111 Additionally, genetic variations (such as polymorphisms) in circadian clock genes can also increase the susceptibility to male reproductive diseases. For instance, Clock and Bmal1 variations can significantly affect semen quality and increase the risk of male infertility.112,113,114 Polymorphisms of Clock and Cry1 are possibly associated with individual susceptibility to abdominal obesity in the Chinese Han population.115 Men with the Cry2 variant C allele were reported to have a significantly increased (1.7 times) risk of PCa.116 The following sections review the roles of circadian clock genes in the occurrence and development of various common male diseases, including LOH, erectile dysfunction (ED), male infertility, and PCa, and explore their potential therapeutic effects in male diseases through circadian rhythm-related pathways (Table 1).

Table 1.

Regulatory mechanisms of the circadian clock on male diseases

Male disease	Regulatory mechanisms related to the circadian clock	Reference
LOH	Reduced expression of genes related to steroid synthesis	34 121 122
ED	NO/cGMP pathway and endothelial dysfunction	127 128 129 130 131 132 133 134
Male infertility	Reduced sperm count, decreased activity of sperm acrosome protein, down-regulated expression of genes involved in spermatogenesis, and impact on the expression of genes related to testicular immune response	41 81 140 141 142 143
PCa	Cell proliferation and apoptosis, and DNA damage responses	150 151 152 153 154

ED: erectile dysfunction; LOH: late-onset hypogonadism; PCa: prostate cancer; NO/cGMP: nitric oxide/cyclic guanosine monophosphate

LOH

The core pathogenesis of LOH is a decrease in testosterone synthesis in Leydig cells caused by aging;117 specifically, key factors were reported to be a reduction in cell quantity and degradation of cell function.118 A clinical study showed that non-standard shift workers with sleep disorders had lower serum testosterone levels and more severe LOH symptoms compared with daytime workers.119 Bremner et al.47 reported that the circadian rhythm of serum testosterone levels was significantly weakened or even absent in healthy elderly men compared with healthy young men. The authors also found that morning testosterone levels were significantly elevated in young men but not in elderly men, and the average testosterone level of healthy elderly men was lower than that of young men throughout the day. Aging has a close relationship with circadian clock genes.120 It has been shown that aging downregulated the expression of a range of circadian clock and steroid synthesis genes and weakened the rhythmic secretion of testosterone in the Leydig cells of rats and mice.34,121 Our earlier research revealed that the traditional Chinese medicine compound “Xiongcan Yishen Formula” upregulated the mRNA and protein levels of circadian clock genes Bmal1, Nr1d1, Per2, and Cry1, as well as steroid synthesis genes Star and Cyp11a1, in testicular tissues of rats with LOH, and increased serum testosterone levels.122 These findings suggest that circadian clock genes may serve as potential targets for the treatment of LOH.

ED

Clinical studies have indicated that changes in the sleep-wake cycle can disrupt the circadian rhythm and impair metabolic homeostasis,17,123,124 thereby affecting the erectile function of men. Pastuszak et al.125 examined 182 males and found that non-standard shift workers had lower International Index of Erectile Function (IIEF) scores (including erectile function, orgasmic function, as well as overall sexual satisfaction). Rodriguez et al.126 recruited 754 males and found that night shift workers had an average IIEF-EF score 7.6 points lower than those who worked during the day time or evening, while non-standard shift workers with sleep disorders had an average IIEF-EF score 2.8 points lower than those without shift. The most common pathogeneses of ED are pathway impairment and endothelial dysfunction. Earlier research suggested that the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway is regulated by multiple circadian clock genes.127,128 NO synthase is a major and essential neurotransmitter in erection,129 and impaired NO production will inevitably lead to vascular ED.130 It was reported that mice with a Per2 mutation exhibited reduced production of NO and vasodilatory prostaglandins, as well as increased release of vasoconstrictors.123,131 Insulin-stimulated NO release was also found to be affected by the Per2 mutation in both active and inactive phases.132,133 Additionally, endothelial dysfunction has been associated with the circadian clock system,127,132 and in Bmal1^−/− mice, endothelial dysfunction and vascular injury were observed.123,134 In summary, the circadian clock system may affect male erectile function by regulating the NO/cGMP pathway and endothelial dysfunction, but the specific mechanisms need to be further clarified.

Male infertility

The causes of male infertility are complex and diverse. Previous studies have shown that circadian rhythm has a significant impact on the reproductive behavior of mammals.135 Although humans are not seasonal breeders, there are still annual rhythmic changes in fertility.135,136,137 Xie et al.135 analyzed 12 245 semen samples and found that the total number and concentration of sperm were higher in spring and lower in summer, but the number of morphologically normal sperm was significantly increased during summer time. Compared with the reproductive season, the expression levels of Bmal1 and Cry1 in the testes of mice were sharply reduced during non-reproductive seasons.138 The temperature fluctuations caused by the adjustment of the light-dark cycle throughout a year may be an important factor explaining the changes in semen quality.135,139 Both the disruption of circadian rhythm and loss of core circadian genes can exert a negative impact on male fertility.140 A study on Egyptian men revealed that night shift work increased the risk of infertility by nearly four times.141 In another study, the average total sperm count of mice after 24 days of circadian rhythm reversal was significantly reduced at all measured time points within 24 h compared with the control group.142 Upon dual knockout of Per1/Per2, the expression levels of genes involved in spermatogenesis (tubulin delta 1) were downregulated, and the spermatogenic function of male mice was markedly reduced.81 Male mice with a Clock Δ19 gene mutation exhibited significantly reduced in vitro sperm fertilization ability and acrosomal enzyme activity.143 The Cry1 knockout led to changes in the expression of genes related to immune response in the testis, alongside increased apoptosis of spermatogenic cells and a reduced sperm count in mice.41 The above research findings suggest that it is important to clarify the interaction between circadian rhythm disorders and male fertility and practice more reasonable lifestyles and work schedules for the treatment and prevention of male infertility.

PCa

Circadian rhythm disorders are closely related to the occurrence of PCa.144,145,146 According to the two epidemiological studies, variations in Bmal1, Per1, and Per3 were associated with an increased risk of PCa.147,148 A recent report also indicated that abnormal expression of Cry2 and Bmal1 was closely related to the progression of PCa.149 Circadian clock genes may be involved in the regulation of several pathways related to PCa, including cell proliferation and apoptosis, as well as DNA damage response.150 A previous study showed that Bmal1 participated in the proliferation of prostate cells by regulating the cell cycle.151 Overexpression of Per1 significantly inhibited the growth of PCa cells while promoting their apoptosis, whereas Per1 levels were downregulated in PCa tissue compared with normal prostate tissue.152 The Nr1d1 agonist (SR9009) was found to significantly suppress the colony formation, cell cycle, and cell migration of PCa cells and promote cell apoptosis. Moreover, Per2 and neuronal PAS domain protein 2 have both been demonstrated to affect the DNA damage response of PCa cells.153,154 The effects of circadian rhythm disorders on the incidence of PCa at the epidemiological and molecular levels suggests that it may be an effective strategy to adjust the timing and dosage of medication based on changes in circadian clock genes in PCa patients to achieve the optimal therapeutic outcome.

Potential treatment methods for male diseases under the framework of circadian rhythmicity

Chronotherapy (i.e., timely drug administration) refers to determining the appropriate drug administration time based on the human circadian rhythmicity to achieve the optimal treatment efficacy and minimize adverse reactions.155 The essence of chronotherapy is to purposefully alter drug levels to match circadian rhythms and optimize drug therapy.156 The application of chronotherapy has been gradually receiving attention in multiple medical fields, such as cardiovascular diseases, oncology, endocrinology, and neurology.157 Chronotherapy is particularly important in the treatment of male diseases. As men age, their testosterone levels and circadian rhythms will change, and such loss of rhythmicity has been associated with the occurrence and development of multiple male diseases.47 Therefore, restoring or simulating the physiological circadian rhythms of testosterone has potential clinical significance for the treatment of male diseases. For instance, Amano et al.11 pointed out that administering low-dose testosterone ointment (Glowmin) in the morning could better simulate the circadian variations of endogenous testosterone and improve symptoms in patients with LOH. This kind of chronotherapy method can improve the treatment efficacy and also reduce patients’ economic burden and time costs. In-depth understanding of the impact of circadian rhythms on the tumor cell cycle also provides a new treatment strategy for the application of chronotherapy in chemotherapy for PCa.158 Research has shown that certain anticancer drugs administered at specific time points can enhance their killing effect on tumor cells while reducing damage to normal tissues.159

In addition to chronotherapy, there are some non-pharmacological treatment options, such as adjusting working hours, phototherapy methods, and intermittent fasting. It was reported that adjusting working hours for night shift workers can help mitigate the impact of night work and light pollution, restore circadian rhythms, and improve the overall health of men.160,161 The use of filtering short wave light may reduce the interference of nighttime light on circadian rhythms, thereby helping men maintain normal hormone levels and physiological function.162 Intermittent fasting has a beneficial effect on metabolic health by regulating the circadian rhythms of the gut microbiome, thereby helping prevent obesity and metabolic syndrome in men.163 In summary, restoring normal circadian rhythms has shown broad prospects in the treatment of male diseases and should be further explored.

CONCLUSIONS AND PROSPECTS

Circadian clock genes are closely related to testosterone synthesis in males. Specifically, by regulating testosterone synthesis in Leydig cells through the central and peripheral circadian clock systems, clock genes can affect a series of physiological activities in the male body. Moreover, circadian rhythm disorders and the abnormal expression of circadian clock genes can induce and exacerbate a variety of male diseases, including LOH, ED, male infertility, and PCa. Therefore, circadian clock genes have attracted increasing attention from researchers, and chronotherapy and other non-pharmacological treatment methods (including adjusting working hours and phototherapy) have shown good application prospects in the prevention and treatment of male diseases. With the increase of aging populations and social industrialization, unhealthy lifestyle habits have posed a significant impact on human health, leading to an upward trend in the development of male diseases. Accordingly, the importance of clock genes in the occurrence and development of male diseases is becoming increasingly prominent.

The current research on the association between circadian clock genes and male diseases has some shortcomings. First, as we discussed in the above section of “Expression of circadian clock genes in testicular tissues,” there is still controversy over whether clock genes exhibit circadian rhythms in testicular tissues. Although a few studies supported the circadian rhythmicity of clock genes in the testis, more researchers failed to observe significant circadian rhythms in the testis as in other organs. This discrepancy may be due to differences in the oscillation amplitude and phase of clock gene expression in different types of cells within the testis. Consequently, during detection of the overall testicular expression level, these oscillations may cancel each other out or cause interference. Therefore, future research should delve deeper into the expression patterns of circadian clock genes in different cell types within the testis, alongside their mutual influences. Innovative techniques, such as the combination of single-cell sequencing and spatial transcriptomics, as well as flow cytometry sorting, can be considered to accurately address the issue of cell heterogeneity in the testis. Second, testosterone synthesis is regulated by the peripheral circadian clock, and by the SCN through the hypothalamic–pituitary–testis axis. However, few studies have explored the steady-state changes of hypothalamic–pituitary–testis axis hormones after the knockout of clock genes or changes in light and dark environments, or whether the circadian rhythm changes of GnRH and testosterone are consistent in the circadian rhythm disruption model. Thirdly, at present, most of the related studies are based on animal models and in vitro experiments. Research into the relationship between circadian clock genes and the regulation of testosterone synthesis in humans is limited to clinical epidemiological investigations and mining and analysis of relevant databases. There is limited direct evidence at the level of a specific mechanism. Moreover, many clinical studies are subjected to small sample sizes, and most studies are cross-sectional, lacking long-term follow-up investigations evaluating the long-term effects of clock gene variations on testosterone levels. These limitations impact the universal applicability and reliability of research results. In addition to these shortcomings, clinical research should conduct multicenter, large-sample studies to clarify the benefits and optimal treatment plan of chronotherapy (i.e., implement testosterone supplementation therapy for related male diseases by following the circadian rhythm of testosterone secretion), and investigate the genetic variations of circadian clock genes and their impact on male health, to provide possible new avenues for early screening and individualized treatment of male diseases. These topics require practical exploration by future clinical researchers.

AUTHOR CONTRIBUTIONS

GN and XZ were responsible for the review design and manuscript writing. BNL was responsible for table creation. HW was responsible for revising the manuscript. RBS, AJP, and HYW participated in the design of the figures. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.