Steroidogenesis is a multistep and dynamic process starting from the peripubertal stages throughout adulthood. The primary organs concerned by steroidogenesis, namely the testes and the adrenal glands, are susceptible to any alteration in this process prior to the completion of puberty [30]. This study aimed to investigate the beneficial impact of naturally-derived silk sericin upon testicular and adrenal steroidogenesis in mice under altered photoperiodism, starting with animals at the peripubertal stages and continuing throughout adulthood. In this context, we followed the previously reported working definitions of ages at stages of postnatal development, according to concentrations of gonadal hormones and performance of social behaviors [31].

Various studies report that disruption of photoperiod affects animal body weight, although the effects may differ among vertebrate groups. It was shown that the field vole (Microtus agrestis) transferred to long-day photoperiod (LD, 16Light: 8Dark) for four weeks increased its body weight by 24.8% compared to animals kept under short photoperiod conditions (SD; 8Light: 16Dark). Interestingly, enlarged testes and seminal vesicles were also found [32]. Also, rats subjected to LD photoperiod increased their body weight compared to animals exposed to SD photoperiod. However, Syrian hamsters react to such conditions in an opposite manner [33, 34]. Similar effects are caused by ALAN, leading to weight gain and obesity in humans, mice, and Australian budgerigars but not in toads Rhinella marina [35, 36]. In agreement with cited studies, in our experiment, LD photoperiod caused a significant increase in the weight of the mice after five weeks. Although the disturbance of the photoperiod did not affect the mass of the testicles, it led to visible histological changes (Fig. 1). This result is consistent with those described by Gouda and Selim [37] and Kus et al. [38]. They demonstrated that exposure to different duration of light has unfavorable effects on the testicular structure in rats. Interestingly, in our experiment, the treatment with sericin restored the typical structure of the seminiferous tubule, the layer of the spermatocytes, the number of Sertoli cells, and the standard structure of Leydig cells compared to disturbed photoperiod groups (Fig. 1).

Reproductive disorders due to circadian rhythm disruption have been less frequently studied in males/men than in females/women. Although it is known that the desynchronization of endogenous circadian clocks affect each level of the HPG axis, the level of sex hormones, the process of spermatogenesis, final sperm concentration and motility, and, ultimately, fertility - both in males of various vertebrate species and in humans [8, 39, 40]. It has been shown that mice exposed to shortened light-dark cycle (Light/Dark = 4 hours/4 hours) for 5 or 10 weeks responded with reduced testes size, abnormal morphology, decreased sperm concentration and motility, and lowered levels of dihydrotestosterone and androstenedione [41]. In our experiment, we found that 5-week exposure to both SD and LD photoperiods reduced sperm count and motility and increased the number of abnormal sperm (Fig. 2). These changes strongly correlated with decreased testosterone levels and, at the same time, increased aromatase levels in this gland (Fig. 3).

Aromatase belongs to the cytochrome P450 family and catalyzes the aromatization of androgens and their conversion to estrogens. The changes in aromatase concentration are season- and age-related. Aromatase is detected in the testicle (Leydig cells or Sertoli cells - depending on age, as well germ cells at each step of spermatogenesis, from gonocytes to spermatozoa). Aromatase is also present in the prostate, as well as also in the brain, the ovaries of females, and cancer tissues [42, 43]. For example, aromatase expression in bank vole testes was more remarkable in animals kept under long photoperiod in which spermatogenesis was fully developed. Thus, its role is substantial and is related to sperm motility properties and its indirect role in the regulation of spermatogenesis [43,44,45,46]. Again, sericin restored testosterone and aromatase levels to levels typical of control animals in our experiment.

The disturbance of the photoperiod also caused an increase in MDA and a decrease in TAC concentration, and a reduction of XO activity (Fig. 4). Such a result indicates that increased oxidative stress is an accompanying phenomenon and probably can contribute to the development of reproductive disorders. To link the effects of photoperiod with disorders of spermatogenesis and oxidative stress, it is worth paying attention to the vital hormone produced by the pineal gland, namely melatonin (Mel). This compound coordinates the master clock and regulates the circadian rhythm and, thus, physiological functions. However, Mel synthesis also occurs in other tissues/organs (e.g., digestive tract, platelets, skin, or bone marrow). Mel exhibits anti-inflammatory and antioxidant activity and is perceived as a reactive oxygen species (ROS) scavenger. Consequently, Mel can protect cells and tissues from infection and oxidative stress [47,48,49]. Rossi et al. [47], who examined men with idiopathic infertility, have clearly proven and transparently presented this relationship. Moreover, male Syrian hamsters and various cell lines showed a negative correlation between Mel concentration in the testes and the expression of proinflammatory factors (TNFα, IL1β, and COX2) as well as a positive correlation between Mel concentration and the expression of antioxidant enzymes such as catalase, superoxide dismutase, and peroxiredoxin. In our experiment, we confirmed that the inappropriate photoperiod in SPG-P and LPG-P groups disturbed the synthesis of Mel, which contributed to the intensification of oxidative stress (and perhaps also proinflammatory effects). The consequence was unfavorable changes in the weight and structure of the gonads and adrenal glands, sperm quantity and quality, hormone concentration, markers of oxidative stress, and cholesterol in the testes and/or serum (Figs. 1-8). Interestingly, such effects were observed not only when the day was extended (LPG-P) but also when it was shortened (SPG-P). This result suggests an even more complex nature of the phenomenon under consideration and encourages further research.

Using natural plant-derived compounds to reduce testicular toxicity caused by various factors is of interest to scientists. Mansour et al. [50] investigated the effects of Ginkgo biloba extract (EGb 761) in reducing methotrexate-induced adverse effects in rat testes. Methotrexate (MTX) is an anti-cancer and immunosuppressant drug with testicular toxicity and infertility side effects. Oxidative stress is generally believed to be involved in the toxicity of MTX. The four-week exposure of rats to MTX led to the development of a complete picture of testicular damage and disorders of spermatogenesis. It was manifested by organ fibrosis and a decrease in the quantity and quality of sperm, as well as in reduction in the level of FSH, LH, testosterone, and reduced glutathione (GSH). Moreover, the concentration of MDA, oxidized glutathione (GSSG), and proinflammatory cytokines were increased. G. biloba extract (EGb-761) effectively reduced oxidative stress and adverse changes in a dose-dependent manner [50]. Similarly, to reduce MTX-induced testicular damage and reproductive disorders in rats, Kamel et al. [51] used the antioxidant properties of ginseng, while Felemban et al. [52] used the ability of amygdalin (Vit B17) to inhibit lipid peroxidation and free radical scavenging. An extract from Gardenia jasminoides Ellis, a genipin, reversed the adverse changes in spermatogenesis and fertility caused by photoperiod disturbance in male mice. A normalization of sex hormones and proteins involved in steroidogenesis was observed, as well as a reduction of atrophy of the seminiferous tubules, decreased vacuolization, and restoration of typical control animals’ sperm motility and concentration [41].

In our study, the vast majority of the adverse effects of circadian disruption were suppressed by the sericin used in the experiment (Figs. 1-8). Sericin constitutes as much as 20–30% of the mass of silkworm cocoons and is a valuable natural protein from the textile industry by-products. Its contemporary use in medicine is related to high biological activity, lack of immunoreactivity, hydrophilicity, a tendency to form a gel, and the ability to create films or scaffolds when combined with other substances. Therefore, it is proposed to use sericin for wound healing, artificial skin, and contact lens production. Silk proteins (mainly Ala, Gly, Ser, Val, and Thr) administered to rats under exercise stress improved physical endurance but also increased testosterone level and sperm count [53]. Consumed sericin has a protective effect on the gastrointestinal tract (anti-tumor, anti-diabetic, anti-constipation properties), and due to its strong affinity to selected drugs, it is considered a drug carrier [22,23,24, 54,55,56]. Moreover, its high antioxidant potential [24, 57,58,59] creates new opportunities for its use wherever oxidative stress occurs, leading to a direct or indirect cause of organism dysfunction. Since reproductive dysfunction due to disturbed photoperiod is associated with increased oxidative stress, thus we postulate that sericin can be a good way of mitigating these adverse effects.