Male infertility is a complex issue with multiple contributing factors, and oxidative stress has emerged as a noteworthy contributor, particularly in the context of VCL and other conditions that lead to testicular hypoxia (Fang, Su et al. 2021). VCL is widely recognized as a common and manageable factor contributing to male infertility, primarily due to its influence on oxidative stress (Birowo, Tendi et al. 2020). The oxidative stress associated with VCL has toxic effects on essential cellular functions, including spermatogenesis, semen production, and the HPG axis. Moreover, the generation of excessive oxidative stress due to VCL can also lead to germ cell apoptosis, further challenging reproductive health (Han, Xiang et al. 2021, Abo El Gheit, Soliman et al. 2022, Wang, Gao et al. 2022). Numerous studies have shown that VCL can induce testicular hypoxia and lead to tissue damage related to the lack of oxygen in the testis (Ameli, Moghimian et al. 2019, Shokoohi, Khaki et al. 2022, Abadi, Boukani et al. 2023).
Antioxidant therapy is identified as a crucial supplementary intervention to mitigate the oxidative damage inflicted upon testicular tissues by VCL. This condition manifests extensive histological damage, leading to the loss of Sertoli and germ cells, ultimately culminating in testicular atrophy. The activation of the intrinsic apoptotic pathway in male germ cells further intensifies the degradation of the testicular tissue (Dolatkhah, Khezri et al. 2022). Nonetheless, encouraging findings propose that the administration of NAC could mitigate these repercussions. Research, exemplified by Babaei et al., underscores NAC's potential to diminish the incidence of germ cell apoptosis, providing optimism for alleviating VCL-induced testicular injuries in rat models (Babaei, Asadpour et al. 2022).
Sperm motility, a critical determinant of successful fertilization, encounters notable impediments in the presence of VCL (Dolatkhah, Khezri et al. 2022). This condition significantly reduces the mobility of sperm on the affected side compared to non-affected counterparts. Sperm movement is intricately linked to the intracellular calcium ion concentration, a process intricately regulated by CatSper channels localized in spermatozoa (Arya, Balasinor and Singh 2022, Shanaz, Hamadani et al. 2022). Studies suggest a correlation between diminished CatSper expression in testicular tissue and a decline in sperm motility (Fallara, Capogrosso et al. 2023).
Our examination through real-time PCR revealed a significant decrease in the expression of Catsper-1 and Catsper-2 genes in the testes of rats subjected to VCL induction. Also, we demonstrated through immunohistochemical examination that the expression of Catsper-1 and Catsper-2 proteins in the testicular tissue decreased in the VCL group.
The findings of our study suggest that NAC treatment resulted in a substantial enhancement in the expression levels of both CatSper-1 and − 2 genes in the animal model. Furthermore, this intervention contributed to a notable increase in the population of motile spermatozoa. The observed improvement in sperm motility following NAC administration can be attributed, at least in part, to the inherent antioxidant properties of this compound. Additionally, the results of the immunohistochemical analysis revealed an increase in the expression of Catsper proteins in the groups treated with NAC. This evidence further supports the potential of NAC as a therapeutic agent in mitigating the detrimental effects of VCL on sperm motility. The underlying mechanism likely involves the influence of NAC on CatSper gene expression, ultimately leading to an enhancement in the quantity of motile spermatozoa (Zhou, Cui et al. 2021).
VCL has detrimental effects on Leydig cells, germinal cells, and Sertoli cells, leading to gradual deterioration in sperm parameters (Neto, Roque and Esteves 2021). This condition induces testicular warming, causing excessive ROS production and hypoxia. The inhibition of enzymes involved in steroid biosynthesis results in reduced intratesticular testosterone (Maheshwari, Muneer et al. 2022). The findings provide rigorous evidence that VCL has significant and direct detrimental impact on key sperm quantity variables. Specifically, VCL was found detrimental to sperm count, motility, and integrity, negatively associated with sperm concentration but not semen concentration. These findings align with observations in oligozoospermic men with VCL, who exhibit reduced testicular size, minimal sperm concentration, and low motility (Maheshwari, Muneer et al. 2022). Increased ROS levels have consistently associated with diminished sperm quality (Poli, Fabi et al. 2022, Shokoohi, Khaki et al. 2022). The research highlights N-acetylcysteine's positive effects may arise from enhanced testicular antioxidant capability. NAC, an antioxidant, prevents extracellular ROS accumulation, hindering caspase-8 activation and the extracellular apoptotic pathway (Pyrgidis, Sokolakis et al. 2021). Therefore, NAC increases motile spermatozoa proportion and improves sperm morphology, enhancing fertilizing competency. Prior research in a testicular torsion rat model also showed N-acetylcysteine's ability to improve Leydig cell steroidogenic capacity and sperm quality, likely through anti-apoptotic effects (Acer-Demir, Mammadov et al. 2020).
Steroidogenic factor 1 (SF-1), a transcription factor, demonstrates early expression in fetal and adult pituitary gonadotropes and Leydig cells in males. This factor plays a primary role in HPG axis development and function. Decreased testosterone may associate with potential SF-1 gene expression defects, but the exact mechanism remains elusive, requiring further investigation (Ameli, Moghimian et al. 2019, Dolatkhah, Khezri et al. 2022).
Various studies have documented a notable elevation in testosterone concentration in male rats subjected to NAC treatment at a dosage of 150 mg/kg over a treatment period (Abedi, Tayefi-Nasrabadi et al. 2023). This observed increase in testosterone levels following NAC administration highlights its potential influence on testosterone regulation, suggesting a promising avenue for therapeutic intervention. The decrease in testosterone serum concentration induced by VCL in rats could potentially result from pathways triggered by LHCGR stimulation, as upon LH binding, it initiates a cascade that stimulates the synthesis of outer mitochondrial membrane (OMM) translocator and steroidogenic acute regulatory (StAR) proteins, which play a crucial role in transporting cholesterol molecules for testosterone production (Ameli, Moghimian et al. 2019, Dolatkhah, Khezri et al. 2022). Additionally, LHCGRs activate supplementary pathways, including protein kinase B, which is essential for Leydig cell survival and proliferation (de Mattos, Pierre and Tremblay 2023). The decrease in testosterone serum concentration observed in rats with VCL could potentially be attributed to pathways triggered by LHCGR stimulation.
VCL exerts a substantial impact on the hypothalamic-pituitary-gonadal (HPG) axis, inducing a decrease in testosterone levels that subsequently leads to reduced secretion of FSH and LH (Dolatkhah, Khezri et al. 2022). However, the exact mechanism underlying this effect remains unclear. In the research, a significant decrease was observed in the expression levels of FSH and LH in the testes of the VCL-induced groups. This reduction in hormone levels might potentially be associated with the rate of SF-1 expression, considering the substantial evidence supporting SF-1's role in regulating genes involved in LH and FSH transcription, along with the GnRH receptor (Dolatkhah, Khezri et al. 2022). Studies involving SF-1-knockout mice demonstrated significantly lower levels of serum LH, indicating a correlation between SF-1 and LH production (Smith, Morin et al. 2023). This LH deficiency can consequently impact testosterone production. The reduced LH levels in VCL-induced rats might result from a failure in SF-1 expression. However, treatment with NAC showed promise in ameliorating the decreased LH levels, potentially by mitigating Leydig cell degeneration and increasing testosterone serum levels.
In relation to testosterone, FSH plays a crucial role in regulating spermatogenesis by binding to its receptor, FSHR, expressed in Sertoli cells (Santi, Crépieux et al. 2020). The reduced germ cell populations observed in the study might be correlated with insufficient FSH or FSHR levels (Santi, Crépieux et al. 2020). The VCL induction activated the extrinsic pathway of apoptosis, leading to an upregulation of inhibin-B in Sertoli cells, which eventually diminished FSHR levels due to excessive ROS production (Bhartiya, Patel et al. 2021, Dolatkhah, Khezri et al. 2022). SF-1 directly influences FSH biosynthesis in the pituitary, regulates FSHR function, and activates protein kinase A, which is central to the FSH signaling pathway in mammalian reproduction (Han, Jiang et al. 2023). Under normal physiological conditions, the presence of SF-1 leads to elevated FSH levels, which in turn stimulate the synthesis of inhibin proteins (Ameli, Moghimian et al. 2019, Han, Jiang et al. 2023). This process then initiates a negative feedback loop, reducing FSH production to maintain hormonal homeostasis. The findings of this study indicate that decreased expression of SF-1 can impair FSHR regulation, consequently diminishing FSH levels within the HPG axis.